Antibacterial activities of the chemical constituents of Schizophyllum commune MST7-3 collected from coal area

Abstract Two fusidane-type active compounds (6 and 7) and five new ones (1–5), along with other nine known compounds (8–16) were isolated from the metabolites of Schizophyllum commune MST7-3. Their structures were elucidated on the basis of spectroscopic analysis. The absolute configurations of compounds 2 and 3 were established by Mosher’s method and optical rotation. Compounds 6 and 7 showed significant antibacterial activities against Stenotrophomonas maltophilia with MIC values of 4 μg/mL and 16 μg/mL, respectively. Graphical Abstract


Introduction
The Gram-negative bacteria has become an issue of great concern worldwide since the 'SKAPE' has been listed as the prime class of opportunistic pathogens to humankind (Tacconelli et al. 2018). This imminent health threat has put humanity in a bind again especially only one new anti-Gram-negative drug was developed from 2000 to 2015 (Butler et al. 2017). It signifies that infections caused by pathogens including resistance-bacteria and superbugs would lead to 10 million deaths per annum and would have an impact on the global economy of more than $100 billion by 2050 (Deng and Yu 2018). Stenotrophomonas maltophilia is an aerobic, gram-negative bacillus that has emerged as an important opportunistic pathogen to clinic infections including meningitis, septicemia, pneumonia, urinary tract infection, skin and soft-tissue infection (Correia et al. 2014;Furuichi et al. 2016;Saggini et al. 2021). Due to inherent resistance to multiple antibiotics of S. maltophilia and lack of antibiotics effective against S. maltophilia, S. maltophilia infection has posed a therapeutic challenge to infectious disease clinicians. Thus, development of new antibiotics has become a high priority in biomedical research. Fungi collected from extreme environments (e.g. acidic, metal-rich, and ultra-high temperatures) have been proved to be a promising source of novel bioactive compounds (Elleuche et al. 2015;Fellet 2017;Sibanda et al. 2017;Stierle et al. 2017), and Penicillium sp. collected from Xinren coal area was demonstrated to produce antibiotic metabolites against Candida albicans in our previous study (Xu et al. 2021). As a continuing effort to discover new antibacterial compounds from the secondary metabolites of fungus collected from coal area, a crude extract of solid-cultured fermentation of Schizophyllum commune MST7-3 displayed medium antibacterial activity against Stenotrophomonas maltophilia (MIC ¼ 2 mg/mL). Two fusidane-type active compounds (6 and 7) and five new ones (1-5, Figure 1), along with other nine known compounds (8-16) were isolated. Here the isolation, structure elucidation, and antibacterial activities of these compounds are described.  displayed a total of 12 carbon signals for one carboxyl (d C 170.3), six aromatic carbons (d C 152.2, 147.6, 124.9, 123.5, 117.5, and 113.2), two olefinic carbons (d C 141.5 and 120.5), two oxymethylenes (d C 67.7 and 66.4), and one methyl (d C 14.0). A 3,4-dioxybenzoic acid fragment was further established by the HMBC correlations from H-2 (d H 7.45) to C-3 (d C 147.6), C-4 (d C 152.2), C-6 (d C 123.5), and C-7 (d C 170.3) (Figures S6 and S43), from H-6 (d H 7.52) to C-2 (d C 117.5), C-4, and C-7, and from H-5 (d H 6.98) to C-1 (d C 124.9), C-3, and C-4. A 4 0 -hydroxy-3 0 -methylbut-2 0 -en-1-yl group was established by HMBC correlations from H 2 -1 0 (d H 4.76) to C-2 0 (d C 120.5), C-3 0 (d C 141.5), from H 2 -4 0 (d H 4.00) to C-3 0 , C-2 0 , and from H 3 -5 0 (d H 1.78) to C-2 0 , C-3 0 , and C-4 0 (d C 67.7). This group was further connected to C-4 via an oxygen-bridge by the HMBC correlations from H 2 -1 0 (d H 4.76) to C-4 (d C 152.2). The configuration of the D 2 0 double bond of 1 was shown to be E by the NOESY correlations of H 2 -1 0 /H 3 -5 0 , and H-2 0 /H 2 -4 0 ( Figure S7). Based on the foregoing data, compound 1 was finally identified as (E)-4-(4-hydroxy-3-methylbut-2-en-1-yl)oxybenzoic acid.

Results and discussion
Compound 2 was obtained as a white amorphous powder. The HR ESIMS and 13 C NMR data were consistent with the molecular formula of C 13 H 18 O 5 , corresponding to five degrees of unsaturation. In the 1 H NMR spectrum, an aromatic AA 0 BB 0 spin sys- .20 and 4.07) and one oxymethine (d H 3.84) were assigned with the aid of HSQC data. The 13 C NMR spectrum of 2 showed 13 signals assignable to six aromatic carbons (d C 162.3, 131.8, 131.8, 114.3, 114.3, and 123.4), one carboxyl (d C 166.9), one ester methoxyl (d C 52.1), and two methyls (d C 26.8 and 25.2). These spectroscopic features were very similar to those of paratrimerin H except for signals for an extra ester methoxy group in 2 (Nguyen et al. 2018). These observations suggested that compound 2 was the methyl ester product of paratrimerin H, which was further confirmed by extensive HMBC correlations analysis (Figures S13 and S43). In particular, the HMBC correlation from H-1 00 (d H 3.89) to C-7 (d C 166.9) secured the location of the methoxyl group at C-7. Therefore, compound 2 was established as methyl 4-(2,3-dihydroxy-3-methylbutoxy)benzoate.

General experimental
High resolution electrospray ionization mass spectra (HR ESIMS) were recorded on a Shimadzu LC MS-IT-TOF mass spectrometer equipped with an ESI interface. Optical rotations were measured on an Autopol I automatic polarimeter. UV spectra were obtained on a Shimadzu UV-2700 spectrometer. IR spectra were recorded by the FT-IR-650 spectrometer (as KBr disk; in cm À1 ). NMR spectra were recorded on a Bruker AVANCE NEO 600 M NMR spectrometer ( 1 H: 600 MHz; 13 C: 150 MHz). Chemical shifts are expressed in d (ppm) referring to the residual solvent peak. Semi-preparative HPLC isolation was performed on a LC 3000 system equipped with UV detector and a Kromasil C 18 column (10 mm Â 250 mm, 5 lm) with flow rate of 3 mL/min. Sample analysis was executed on LC-10Atvp system equipped with SPD-10Avp detector and CHIRALPAKV R IA chiral column (Lot No. IA00CE-UF073, 4.6 mm Â 250 mm, 5 lm). Sephadex LH-20 was purchased from GE healthcare company. Silica gel (200-300 mesh) for column chromatography (CC) and silica gel GF 254 (10-40 lm) for TLC and preparative TLC were obtained from Qingdao Haiyang Chemical Co. Ltd., China. All solvents were of analytical grade.

Fungal material
Schizophyllum commune MST7-3 was isolated from the soil collected in Xinren coal area of Guizhou province in China, in May 2020, and identified on the basis of the sequence analysis of the ITS region of the rDNA (see Supplementary material S1). The fungus was deposited at the Microbiological Collection Center of Guizhou Medical University (GMU-2020-MST 7-3).

Antibacterial assay
The antibacterial activity was evaluated according to the reported procedure (Liu et al. 2020) with minor modification. All test microorganisms were prepared using a final concentration of 5 Â 10 5 CFU/mL for antibacterial assay. Specifically, strains of E. coli ATCC 25922, P. aeruginosa ATCC9027 and E. cloacae ATCC13047 were incubated in Mueller-Hinton Broth (Lot: 401E031, Solarbio) at 37 C, S. maltophilia ATCC 13637, K. pneumonia CGMCC 1.839 and A. hydrophila ATCC 7966 were cultured on 0002 medium (1% peptone, 0.3% beef extract, 0.5% NaCl in distilled water with pH 7.0) at 30 C, V. vulnificus NH 87-17 and V. alginolyticus ATCC17749 were grew on 2216E Liquid Medium (Lot: 20210529, Hopebio) at 30 C and 37 C, respectively. V. parahaemolyticus ATCC 17802 was incubated in 0232 medium (1% peptone, 0.3% beef extract, 3% NaCl in distilled water with pH 7.0) at 37 C. Then two-fold dilution of samples (1-16) were made in 96-well plates (128 lg/mL and 2 lg/mL for the starting and the end concentrations, respectively, 100 lL in each well), and 100 lL of bacterial suspension was transferred to the wells to incubate for 16 h at their optimum culture temperatures. The final concentration of 0.25% TTC (2,3,5-triphenyl tetrazolium chloride, Sigma) in wells as microorganism growth indictor was added and the microorganism was continuously incubated for 3 h before MIC reading. Equivalent amounts of DMSO (2.5%) and medium were successively used as negative controls and blank control. Trimethoprim and levofloxacin were used as positive controls. All experiments were performed in three replicates. The growth curve of S. maltophilia was determined according to literature . Briefly, 100 lL of bacterial suspension (5 Â 10 5 CFU/mL) and samples (blank, DMSO, and compound 6) were successively added into MH culture solution and incubated at 37 C, and the growth was tested by measuring OD 600nm using microplate reader at intervals from 0 to 36 h. All experiments were performed in triplicate and each with 4 parallels. The data were expressed as the mean ± SD and analyzed through ANOVA by using Graphpad prism 9.0.

Disclosure statement
No potential conflict of interest was reported by the authors.