Test strain, chemicals and media

The strain of bacteria were tested: Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 which were obtained from Guangdong culture collection center. The genomic DNA isolation kit was purchased from Takara (Beijing, China). Microbiological media were purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd (GuangZhou, China). High-performance liquid chromatography (HPLC) grade methanol was purchased from Honeywell. Solvents used for extraction and column chromatography were of analytical grade which were obtained from Guangdong Guanghua Sci-Tech Co., Ltd (GuangZhou, China).

Primary antimicrobial activity assay

Primary screening for antimicrobial activity was performed by growing cultures on Luria-Bertan (LB) agar plates swabbed with the MRSA suspension (1–5 × 104 CFU/mL). 100 µL of the ethyl acetate extract obtained from strain WA10-1-8 was added to an oxford cup on the LB agar plates and the incubated at 28 ℃ for 12–14 h. 100 µL of the vancomycin was used as positive control with the concentration of 128 µg/mL and methanol was used as a negative control. The experiment was repeated in triplicate for each test strain to record the average diameter of the inhibition zone [21].

Isolation and identification of S. Albovinaceus WA10-1-8

The P. americana samples collected from the wild were rinsed with running water, soaked in 75% ethanol for 2 min, and then disinfected with 3.5% sodium hypochlorite for 2 min. Afterward, they were washed three times with sterile water and dried with sterile absorbent paper. The abdomen of the P. americana was dissected using a scalpel, and its intestines were removed. The intestines were placed in a homogenizer with sterile water for grinding, and then diluted to the desired concentrations (10 mg/L, 0.005 mg/L, 0.0025 mg/L). Next, samples at different dilution concentrations were taken and subjected to separation using the plate dilution method and streak plate method. Each dilution concentration was repeated three times and incubated in a 28 ℃ incubator for 3–7 d until the colonies grow. Colonies with distinct characteristics were selected and further purified on NA plates (nutrient agar medium). The purified colonies were stored on slant ISP-2 medium for preservation.

S. albovinaceus WA10-1-8 was inoculated on Gause’s synthetic agar no.1 and cultured in constant-temperature incubator at 28 ℃ for 4–5 d, during which time the culture had entered the logarithmic growth phase. Streptomyces strain WA10-1-8 was preliminarily identified based on colony morphological characteristics on Gause’s synthetic agar no.1 and gram staining characteristics under microscope. Whole genomic DNA of strain WA10-1-8 was extracted using the GenElute Bacterial Genomic DNA Kit (Sigma) according to manufacturer’s instructions. After that, used universal bacterial primer set 27 F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-TAC GGC TAC CTT GTT ACG ACT T-3′) to amplify 16 S rRNA gene [22]. PCR was performed in a total volume 50 µL reaction containing of 1 µL DNA template, 1 µL upstream primer (Invitrogen), 1 µL downstream primer (Invitrogen), 25 µL PCR Premix (Invitrogen) and 22 µL sterile ddH2O. Thermocycler conditions began with an initial denaturation step at 94 °C for 4 min followed by 35 cycles each consisting of 30 s at 94 °C, 30 s at 55 °C for annealing, and 30 s at 72 °C for extension followed by a post-cycle extension at 72 °C for 5 min. The resulting PCR products were verified by electrophoresis on a 1.5% agarose gel and was sequenced by BGI Genomics Institute (GuangDong, China). The sequenced 16 S rRNA sequence was uploaded to the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/), and used BLAST software to compare all sequences available in GenBank [23]. Sequences were aligned with Clustal software and MEGA 11 was used to carry out a phylogenetic tree of the alignment using the maximum likelihood method [24].

Detection of anti-MRSA activity and HPLC analysis of metabolites from S. Albovinaceus WA10-1-8

Metabolite containing maximum content of streptophenazines and optimal anti-MRSA activity are obtained by changing fermentation temperature. We evaluated the effect of culture temperature from 24 ℃ − 38 ℃ on the change of streptophenazines in metabolites.

At the end of the fermentation cycle the mycelium and supernatant was separated by centrifugation at 8,000 rpm for 15 min. Collection of supernatant and extraction of three times with equal volume of ethyl acetate to obtain fermented crude extract. Finally, The anti-MRSA activity (MRSA ATCC 43300) was assessed using the crude extract, and crude extracts were determined by high-performance liquid chromatography (HPLC). The samples were analyzed triplicate by analytical HPLC after filtering use 0.22 μm syringe filter. The injection volume was set at 10 µL and the chromatographic method was constituted by a gradient of mixtures of solvents A (0.1% acetic acid in H2O) and B (methanol) of: 0–5 min (10% B); 5–40 min (10–100% B); 40–50 min(100% B); 50–51 min (100 − 10% B); 51–60 min (10% B).

Fermentation, extraction, isolation of streptophenazine compounds

S. albovinaceus WA10-1-8 a single colony inoculated in 100 mL ISP1 liquid medium and cultivated as seed medium in 28 °C, 180 rpm thermostatic shaker for 2 d. RA medium was used as the fermentation medium: maltose extract (10 g/L), glucose (10 g/L), maltose (10 g/L), corn syrup (5 g/L), soluble starch (20 g/L), calcium carbonate (2 g/L) and trace elements (100 µL/L). 5% of seed medium was transferred into 500 mL Erlenmeyer flask containing 300 mL of RA medium as production flasks and incubated at 34 °C and 180 rpm for 15 d. The total 7 g crude extract was obtained from 72 L of fermentation, which were extracted with an equal volume of ethyl acetate.

The chromatography separation of the fermentation crude extract was performed using a silica gel column (50-cm height×4.5-cm diameter) with 200 g of 200–300 mesh silica gel powder as the stationary phase. The separation was carried out using petroleum ether-ethyl acetate systems (9:1, 8:2, 7:3, 6:4 v/v) and dichloromethane-methanol systems (9:1, 8:2, 7:3, 6:4 v/v) for gradient elution, with each solvent system eluted for 5 column volumes (1500 mL). All fractions from the silica gel column were pooled based on thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis. The characteristic chromatographic absorption peaks of streptophenazine were detected in pooled fractions 2, 3, and 7.

Fraction 2 was separated using a Sephadex LH-20 column (70-cm height×3-cm diameter) with approximately 1000 mL of 100% methanol, and collected the eluted fractions. Fraction Fr-2-2 contained the target compound, and was further separated using Sephadex LH-20 column with approximately 600 mL of 100% methanol elution, yielding fractions Fr-2-2-1 and Fr-2-2-2. Fraction Fr-2-2-1 was purified by semi-preparative HPLC to obtain compound 1 (15.3 mg) and compound 2 (11 mg).

Fraction 3 was separated using a Sephadex LH-20 column (70-cm height×3-cm diameter) with approximately 1000 mL of 100% methanol for isocratic elution, yielding fractions Fr-3-1 ~ Fr-3-3. Fr-3-2 and Fr-3-3 contained the target compound. Fr-3-2 was purified by semi-preparative HPLC to obtain compound 3 (7.5 mg). Fr-3-3 was further separated using a Sephadex LH-20 column (70-cm height×3-cm diameter) with approximately 350 mL of 100% methanol for isocratic elution, yielding fractions Fr-3-3-1 and Fr-3-3-2. Fr-3-3-2 was purified by semi-preparative HPLC to obtain compound 4 (7.0 mg).

Fraction 7 was separated using a Sephadex LH-20 column (70-cm height×3-cm diameter) with approximately 1200 mL of 100% methanol for isocratic elution. Fraction Fr-7-2 was further separated using a Sephadex LH-20 column (40-cm height×2.5-cm diameter ) with approximately 200 mL of 100% methanol for isocratic elution, yielding fractions Fr-7-2-1 and Fr-7-2-2. Fraction Fr-7-2-1 was purified by semi-preparative HPLC to obtain compound 5 (5.5 mg), and Fr-7-5 was purified by semi-preparative HPLC to obtain compound 6 (4.0 mg).

The semi-preparative HPLC conditions for the purification of compounds 1–4 were as follows: a methanol-water system (60%:40%) as the mobile phase, with a sample injection volume of 100 µL, a flow rate of 2.5 mL/min, a column temperature of 25 °C, detection at dual wavelengths of 250 nm and 360 nm, and a YMC-Pack ODS-AQ 250 × 10.0 mm I.D., S-5 μm, 12 nm column type. The semi-preparative HPLC conditions for the purification of compounds 5–6 were as follows: a methanol-water system (30%:70%) as the mobile phase, with a sample injection volume of 100 µL, a flow rate of 2.5 mL/min, a column temperature of 25 °C, detection at dual wavelengths of 250 nm and 360 nm, and a YMC-Pack ODS-AQ 250 × 10.0 mm I.D., S-5 μm, 12 nm column type.

Streptophenazine T (6): yellow solid, soluble in methanol, DMSO and water; [α]20D −23.35 (c 0.01, MeOH); UV(MeOH) λmax (logε) 205(4.56), 251(4.76), 348(3.91), 365(4.08) nm; IR (KBr) νmax 3304, 2943, 2831, 1448, 1409, 1107, 1022 cm− 1; ECD (10 mg/L, MeOH) λmax (Δε) 251 (− 6.3) nm. 1H NMR (CH3OH-d4, 500 MHz) and 13C NMR (CH3OH-d4, 500 MHz) see Table 1; HRESIMS m/z 427.18582 [M + H]+ (calcd for C23H27N2O6: 427.17944).

GNPS Molecular networking analysis and MS/MS spectra annotation

Using the Global Natural Product Social Molecular Networking (GNPS) platform (https://gnps.ucsd.edu) to construct a molecular network [25]. The MS2 data of crude extracts and solvents (blank) were converted into mzXML file format using the MSConvertGUI tool and uploaded to the GNPS platform. The precursor ion mass tolerance was set to 0.02 Da, the fragment ion tolerance was set to 0.02 Da, and the cosine score was 0.7, with a maximum molecular family size of 100 for the construction of the molecular network (MN). The molecular network was visualized as a node and edge network using Cytoscape (3.9.1) [26]. Node sizes and edge thicknesses were automatically adjusted according to precursor ion abundance and the relationships between precursor ions, with redundant nodes manually removed. Additionally, various databases and tools were used for the manual annotation of spectra of interest, including Pubchem, Sci-finder, and Competitive Fragmentation Modeling for Metabolite Identification (CFM-ID, https://cfmid.wishartlab.com/) [27].

Spectroscopic analysis

The detection of HRESIMS and HRESIMS/MS was performed using a Thermo Scientific Ultimate 3000 system connected to a Q Exactive Orbitrap detector, equipped with a Hypersil Gold C18 column (100 × 2.1 mm, 1.9 μm). The crude extract was dissolved and diluted to 1 mg/mL in chromatographic methanol. The solvent system consisted of 0.1% acetic acid solution (A) and methanol (B). The gradient program was: 0–5 min (10% B), 5–40 min (10–100% B), 40–50 min (100% B). All 1D and 2D NMR spectra were obtained using a Bruker AVANCE III 500 MHz NMR spectrometer (Bruker Company, Germany). Analytical HPLC was performed on a Waters 2695–2996 high-performance liquid chromatography system, using a reverse-phase YMC-Pack ODS-AQ column (250 × 4.6 mm, 5 μm, YMC Company, Japan). Semi-preparative HPLC was performed on a Waters high-performance liquid chromatography system, using a YMC-Pack ODS-AQ column (250 × 10.0 mm, 5 μm, YMC Company, Japan).

Minimum inhibitory concentration (MIC) testing

The minimum inhibitory concentration (MIC) of streptophenazine compounds against MRSA ATCC43300 was measured using the broth dilution method [28]. In this experiment, MRSA was cultured in LB broth at 37 °C until the cell density reached approximately 1–5 × 106 CFU/ml. Streptophenazine compounds were tested in the concentration range of 0–128 µg/ml. The anti-MRSA activity was observed after 12 h of incubation at 37 °C. The lowest concentration of the compound that displayed antibacterial activity was recorded as the MIC value. The experiment was repeated three times, with vancomycin used as a positive control.

Scanning electron microscopy analysis

The tested bacterial suspension (1.5 × 105 CFU/mL) were treated with 1×MIC of the compounds for 12 h at 37 °C. The supernatant was removed by centrifuged and then the tested strain were placed on a cover glass and fixed overnight with 2.5% glutaraldehyde at 4 ℃. Fixed samples were washed with 1×PBS three times for 20 min each and dehydrated in increasing concentrations of ethanol (20, 40, 60, 80, and 100%). The coverslips were finally dried then analyzed by Quanta FEG 200 FESEM at an accelerating voltage of 2–19 kV under standard operating conditions.