MEZ6 was found in the soil of Meiyuan, Central China Normal University. The strain was cultivated at 30°C for 2 d in TY medium (3 g/L yeast extract, 5 g/L tryptone and 1.3 g/L CaCl₂·6H₂O, pH 7.0) (Zhao et al., 2022). Professor Peng Jian’s department at Guizhou Medical University provided MRSA. MRSA was cultured in TSB media (15 g/L tryptone, 3 g/L soybean papain hydrolysate, 5 g/L NaCl, 2.5 g/L glucose, and 2.5 g/L KOH, pH 7.2) at 37°C (Peng et al., 2021). The remaining strains (Pseudomonas aeruginosa CMCC 10104, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606, Klebsiella pneumoniae ATCC 700603, Cryptococcus neoformans H99, Candida albicans ATCC 10231, and Aspergillus flavus 3357) were stored in the laboratory and cultured in SDB media (10 g/L tryptone, 5 g/L glucose, pH 7.2) at 30°C for 2 d.

The bacteriostatic zone method was used to determine the antibacterial activity of each strain. The tested strains were evenly coated on the plate, and after drying, 20 μL of the MEZ6 bacterial mixture was applied to the plate. As a negative control, the same volume of water was used. The mixture was then placed in an incubator at 37°C for 24 h for observation. Antibacterial activity is measured by the diameter of the antibacterial zone (Li et al., 2018).

For co-inoculations via different culture methods, a 2-day-old MEZ6 culture in TY medium was centrifuged at 10,000 rpm for 6 min, and the supernatant was filtered through a 0.22 μm filter. This 200 μL filtrate was inoculated into a MRSA culture (MRSA final concentration of 1 × 10⁶ CFU/mL). For cell lysate preparation, MEZ6 cells were sonicated, and the lysate was centrifuged to remove cell debris. The resulting supernatant was then added to the MRSA culture (final concentration of 1 × 10⁶ CFU/mL), and an equal volume of TSB medium was added as a control (Zhao et al., 2022).

The minimum inhibitory concentration (MIC) was measured using broth microdilution according to the criteria of the American Society for Clinical and Laboratory Standards Institute (CLSI) criteria (Cuenca-Estrella et al., 2010). The strains were grown to the exponential phase and washed three times with PBS (10 mM, pH 7.4). Then, 100 μL of the MRSA suspension (1 × 10⁶ CFU/mL) was inoculated into a 96-well plate, followed by the addition of 100 μL of a serially diluted MEZ6 antimicrobial material mixture, and the plate was subsequently incubated at 37°C for 24 h. The OD_600nm values of each well were measured via a microplate reader. The range of MEZ6 antimicrobial material concentrations evaluated was 0.2–80 mg/mL, and PBS and medium were used as negative and blank controls, respectively. The well containing the bacterial culture without drug treatment served as the 100% growth control, and the minimum drug concentration that resulted in a 50% reduction in absorbance was determined as the MIC value.

To study the effects of secondary metabolites on MRSA, bacterial suspensions (1 × 10⁶ CFU/mL) were treated with final concentrations of 2 × MIC or PBS for 2 h. The bacteria were rinsed twice with PBS before being fixed with 2.5% glutaraldehyde at 4°C overnight. The bacteria were then dehydrated in various concentrations of ethanol. A bacterial suspension free of MRSA was utilized as a control for comparison. Images were acquired with a Hitachi Regulus SU8100 (Tokyo, Japan).

Propidium iodide (PI, 20 μM) was used as a probe to analyze changes in the cell membrane permeability of MRSA after treatment with different concentrations of the antimicrobial substance MEZ6. The final concentration of 5 μM PI was incubated with the bacterial suspension (1 × 10⁶ CFU/mL) for 10 min, followed by treatment with MEZ6 metabolites (2.5–20 mg/mL) at 37°C for 1 h, with PBS used as a control. The fluorescence value of the bacteria was subsequently measured using a microplate reader (BioTek SYNERGY-H, Agilent, USA), with excitation and emission wavelengths set at 535 nm and 615 nm, respectively (Fadhel Abbas Albaayit et al., 2022).

The fluorescence probe 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the intracellular generation of ROS. The bacterial suspension (1.0 × 10⁶ CFU/mL) was mixed with DCFH-DA (final concentration of 10 mM) and incubated at 37°C for 30 min in the dark. After incubation, the suspensions were treated with MEZ6 metabolites (1/2, 1, 2, or 4 × MIC) at the indicated concentrations for 1 h at 37°C. The fluorescence intensity was evaluated using a multifunctional fluorescent enzyme marker with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. N-acetylcysteine (NAC) at a concentration of 6 mM was used as a control agent to quench the production of reactive oxygen species, while PBS served as a negative control.

The effect of MEZ6 antimicrobial material on MRSA biofilm formation has been previously tested and described (Peng et al., 2021). An XTT test kit (Shanghai, BeatBio) was used to assess the absorbance at 490 nm via a multifunctional microplate reader after treatment with various doses of the antibacterial agent. Moreover, we used 0.5% crystal violet to measure the total biomass of biofilms at 560 nm (Vazquez-Armenta et al., 2024), with PBS serving as a negative control.

The experiment involved streaking MRSA strains stored at −80°C onto TSA plates and incubating them at 37°C for 1 d. MRSA strains were treated with 0, 1/2 MIC, MIC or the 4 MIC of MEZ6 secondary metabolites for 4 h, after which the supernatant was centrifuged. A UV spectrophotometer was then used to measure the absorbance at 260 nm (Hsu et al., 2005). The experiment was conducted in three parallel groups and repeated three times to ensure that the results were reliable and reproducible.

MRSA cultures were cultured in TSB overnight to assess the influence of secondary metabolites on bacterial virulence genes. The exponential phase culture was collected when the OD_600nm reached approximately 0.4. The cells were washed with PBS and then subjected to 1/2 × MIC for 4 h. RNA was extracted using a RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA synthesis and quantitative reverse transcription (RT)-PCR were performed according to the manufacturer’s instructions (Bio-Rad, CA, USA), with the primers provided in Table 1. The qPCR cycling settings were 95°C for 30 s, 40 cycles of 95°C for 5 s, and 55°C for 30 s, followed by melt curve analysis from 65 to 95°C.

The bacteria were picked from fresh plates and incubated in 50 mL of TY liquid medium at 200 rpm/min and 30°C for 2 d. The 1% seed mixture was incubated in 1 L of fermentation medium (5 g/L yeast extract, 1 g/L tryptone, 5 g/L glucose, 5 g/L beef extract and 5 g/L NaCl) for 2–3 d at 200 rpm/min and 30°C (Zhao et al., 2022). After centrifugation at 10,000 rpm for 10 min, the mixture was collected and filtered through a 0.22 μm filter.

A macroporous adsorption resin (Mitsubishi DIAION type 80*800 mm) was filled and then pretreated with ethanol and purified water, followed by fermentation broth. The samples were subsequently washed with 3 volumes of filtered water before being eluted with 50% ethanol to a light color (approximately 3 volumes) and 95% ethanol to light color (approximately 3 volumes). The 50 and 95% ethanol eluents were mixed, concentrated, and dried under decreased pressure before being separated and purified via reverse-phase separation. Then, pure chemicals were used in antimicrobial studies.

To enrich the macroporous resin, 1/5 of it was dissolved in 20 mL of 50% acetonitrile, filtered through a 0.45 μm membrane, and then injected. Mobile phase A was acetonitrile, while mobile phase B was water containing 0.1% acetic acid. The peaks with the highest purity in the crude product were collected separately and tested via HPLC. The coarse separation process described above was repeated five times. The enriched macroporous resin is coarsely separated, and then the matching flow content is merged based on the HPLC results, concentrated, and dried under reduced pressure before the target is further purified. For the elution methods, see Supplementary Table S1.

For injection, 1/3 of the crude target was dissolved in 12 mL of 40% acetonitrile aqueous solution and filtered through a 0.45 μm filter membrane. Mobile phases A and B contained acetonitrile and 0.1% acetic acid in water, respectively. The flow was collected into separate bottles and examined via HPLC. The above purification process was repeated three times, resulting in a purity of more than 95% for the target stream. Under reduced pressure, acetonitrile was removed from the target material with a purity greater than 95%. The target was then freeze-dried to provide a target with certified purity. Antibacterial tests were performed on the isolated and purified compounds. The elution methods are shown in Supplementary Table S2.

Antibacterial tests were performed on the isolated and purified compounds. Agar well diffusion was employed to examine the antibacterial activity of the extract. MEZ6 was used as a standard. One hundred microliters (1 × 10⁶ CFU/mL) of MRSA were spread evenly on agar plates. Diapers (6 mm) were created on the plates, and 100 μL of different isolated and purified compounds were loaded. The plates were incubated at 37°C for 24 h. After incubation, the apparent inhibition zone provides insight into the antibacterial potential of the isolated and purified compounds (Li et al., 2018).

The compound’s structure was determined using one-dimensional nuclear magnetic resonance (1H-NMR, 13C-NMR), two-dimensional nuclear magnetic resonance (2D-NMR), high-resolution mass spectrometry (HR-ESI-MS), Marfey’s analysis, and reference literature studies.

A checkerboard assay was used to evaluate the synergistic effects of the purified MEZ6 compounds with conventional antibacterial agents against MRSA (Yan and Hancock, 2001). Briefly, a 4 × 6 matrix was formed in a 96-well microtiter plate with twofold serial dilutions of MEZ6’s purified compounds combined with L-tryptophan (L-Trp). The fractional inhibition concentration index (FICI) was calculated as follows: FICI = MIC of compound A in combination/MIC of compound A alone +MIC of compound B in combination/MIC of compound B alone. Synergy, FICI ≤ 0.5; additive effect, 0.5 < FICI ≤ 4; antagonism, FICI > 4.

Every experiment was performed at least three times. Student’s t-test was used for statistical analysis, and p < 0.05 was considered a significant difference.

MEZ6 demonstrated a substantial inhibition zone against MRSA (Supplementary Figure S1), with an inhibition rate of up to 98.98% (Table 2). In contrast, there were negligible antibacterial activity on the gram-negative bacteria or fungi examined. To further evaluate the antibacterial mechanism of MEZ6 against MRSA, antimicrobial studies were carried out using both the cell lysate and the culture supernatant. The results revealed that the active compounds preventing MRSA growth were largely found in the supernatant (Figure 1). Moreover, the minimum inhibitory concentration (MIC) of MEZ6 secondary metabolites was 5 mg/mL, and the vancomycin MIC was 2 μg/mL.

The antibiofilm potential of the MEZ6 secondary metabolites was evaluated using various methods. The XTT assay was performed to assess MRSA survival following treatment with MEZ6 secondary metabolites. At the highest concentration of 4 MIC (20 mg/mL), a significant reduction was observed (Figure 2A), and showing a dose-dependent effect on the MEZ6 secondary metabolite concentration. The results of crystal violet staining revealed that the highest concentration of MEZ6 secondary metabolites 4 MIC (20 mg/mL) significantly reduced the total biomass of MRSA biofilms (Figure 2B).

It is well known that virulence factors allow MRSA to withstand the host immune system and exacerbate infections. A thorough examination of these virulence variables will provide important insights into MRSA antibiotic resistance. The expression levels of MRSA virulence genes were detected via real-time quantitative PCR following treatment with various doses of MEZ6 secondary metabolites. Figure 2C shows that a 1/2 × MIC treatment for 4 h reduced the expression of virulence genes, including accessory gene regulator A (agrA), cytokine-like factor 1 (clf-1), and immunoglobulin G-binding protein A (spa).

Propidium iodide (PI) cannot permeate the intact membranes of live cells. However, necrotic cells, which have lost membrane integrity, allow PI to enter and attach to DNA. Based on this feature, PI labelling was employed in this work to distinguish between dead and live cells. Figure 3A shows that when the secondary metabolite concentration increased, so did the number of dead cells. The influx of PI indicates that MEZ6 secondary metabolites may cause damage to the MRSA cell membrane, impair membrane integrity, and increase membrane permeability. The amount of cell membrane damage was proportional to the concentration of MEZ6 secondary metabolites, indicating a concentration-dependent effect.

Reactive oxygen species (ROS) generally maintain low concentration levels within the cell, and the accumulation of higher concentrations of ROS can damage cellular structures. As shown in Figure 3B, ROS accumulation in bacteria increased significantly after treatment with MEZ6 secondary metabolites. Furthermore, the addition of the antioxidant N-acetyl-cysteine (NAC) at a concentration of 6 mM reduced the production of ROS, as shown in Figure 3B. These results suggest that the presence of MEZ6 secondary metabolites induces ROS production and may plays a key role in the antimicrobial effects exerted by MEZ6 secondary metabolites.

This study used the DNA concentration in the MRSA culture as a reference to determine the DNA content in the MRSA culture following treatment with various amounts of MEZ6 secondary metabolites. Figure 3C shows that as the quantity of secondary metabolites increased, so did the DNA content in the MRSA culture, indicating a concentration-dependent effect.

To study the impact of MEZ6 and its metabolites on the integrity of the MRSA cell wall and cell membrane, SEM was used to examine the morphological and ultrastructural changes in MRSA cells after 4 h of treatment with MEZ6 and its secondary metabolites.

Figure 4 shows that the MRSA cells in the control group were clear and undamaged, with a rather smooth surface (Figure 4A). In contrast, when MRSA cells were co-cultured with a 1 × MIC (5 mg/mL) of the metabolites, several cells broke, releasing their contents. The cell surface roughened, and cell aggregation occurred (Figure 4B).

First, the antimicrobial activity of the crude extract was tested, and the results revealed that the 100% resin extract had significant antibacterial activity (Figure 5A). The fraction exhibiting antibacterial activity was further isolated and purified, resulting in seven fractions. After their antibacterial activities were validated, it was discovered that the sixth fraction created a distinctive inhibitory zone (Figure 5B). This purified fraction was further processed, yielding six pure chemicals (Figure 5C). One of these compounds had a prominent inhibitory zone, and after further purification, a pure sample was produced (Figure 5D).

It reveals that TAF may serve as a key structural component in MEZ6 secondary metabolites through mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis (Figure 6A, Supplementary Figure S2–S7). We compared the pure chemical we obtained to a commercially available reference via high-performance liquid chromatography (HPLC) (Figure 6B). The results revealed that the retention periods were constant and that the peaks were solitary. We determined the minimum inhibitory concentration (MIC) of the TAF to be 3.3 mg/mL (Supplementary Figure S8). We also found that when MEZ6 pure products were combined with L-tryptophan against MRSA, the FICI value was 1.15 (0.5 < FIC ≤ 4), which indicated an additive effect (Supplementary Figure S9).