Chemical constituents of Aspergillus udagawae isolated from the soil of the Xingren coal areas and their antibacterial activities

Abstract A new helvolic acid derivative (1), together with nine known compounds (2-10) were isolated from the metabolites of Aspergillus udagawae MST1-10 with the bioassay-guided fractionation method. Their structures were identified on the basis of spectroscopic analysis. The absolute configuration of compound 1 was elucidated through NOESY and ECD spectra. Compound 2 displayed significant antibacterial activities against Stenotrophomonas maltophilia with MIC value of 2 μg/mL (Trimethoprim, MIC = 64 μg/mL), and with biofilm inhibition rates of 96.41%, 87.77%, and 41.70% at 4MIC, 2MIC, and MIC, respectively. Graphical Abstract


Introduction
Multidrug-resistant organisms, especially Gram-negative pathogens, are prominently emerging worldwide and lead to an increasing morbidity and mortality (Looney et al. 2009;Jones et al. 2022). Stenotrophomonas maltophilia is an aerobic Gram-negative opportunistic pathogen which was ranked third among the most common non-fermenting Gram-negative bacillus (Singhal et al. 2017;Baseri et al. 2021). Moreover, S. maltophilia is intrinsically resistant to many antibiotics including carbapenems (Brooke 2012;Azimi et al. 2020;Baseri et al. 2021). Due to lack of antibiotics effective against multidrug-resistant organisms, development of new antibiotic has become an urgent task in pharmaceutical research. Fungi are an important resource for the discovery of new antibiotics since it produced a huge number and variety of secondary metabolites with broad biological activities (Demain 2014;Elsbaey et al. 2022;Hawas et al. 2022). As an ongoing effort to explore new antibiotics or their leading compounds from the secondary metabolites of fungi (Xu et al. 2014;Wang et al. 2022), a strain of Aspergillus udagawae collected from Xingren coal areas was shown to produce secondary metabolites with promising antibacterial activity against S. maltophilia (MIC ¼ 1 mg/mL). Whereafter, a new helvolic acid derivative (1), along with nine known compounds (2-10) were isolated. Here the isolation, structure elucidation, and antibacterial activities of these compounds are described.

Results and discussion
The cultured medium of A. udagawae was extracted with EtOAc and further subjected to a silica gel column chromatography using bioassay-guided fractionation method. Thus, a new compound (1, Figure S1) and nine known compounds (2-10) were isolated from the antibacterial fraction.
Inspirited by the antibacterial activity of crude extract against S. maltophilia, other eight strains of Gram-negative pathogenic bacteria were selected for antibacterial assay. All isolated compounds 1-10 were evaluated for antibacterial activities against Acinetobacter baumannii ATCC 19606, Aeromonas hydrophila ATCC 7966, Escherichia coli ATCC 25922, Klebsiella pneumoniae CGMCC 1.839, Pseudomonas aeruginosa ATCC9027, Stenotrophomonas maltophilia ATCC 13637, Vibrio vulnificus NH 87-17, Vibrio alginolyticus ATCC17749, and Vibrio parahaemolyticus ATCC 17802. Compounds 2-7 showed antibacterial activities against S. maltophilia ATCC 13637 with MICs of 2, 4, 8, 64, 16, and 128 lg/mL, respectively (Trimethoprim, MIC ¼ 64 lg/mL). No inhibitory activity was observed for the rest of pathogenic bacteria even at high concentration of 128 lg/mL. The bacterial growth curve of 2 displayed S. maltophilia was significantly suppressed at the concentrations from 2MIC to 8MIC within 24 hours ( Figure S13). While microbial biofilm formation is considered to be a kind of life phenomenon for bacteria to adapt to the natural environment (Flemming et al. 2016). Herein, biofilm inhibition assay was further conducted to study the antibiofilm effect of compound 2. The existing biofilms treated with different samples were quantified by crystal violet staining. Comparing with natural group, experimental groups exhibited significant biofilm inhibition rates of 96.41%, 87.77%, and 41.70% at 4MIC, 2MIC, and MIC, respectively ( Figure S14). This results suggested the antibacterial activity of 2 was related to the inhibition of biofilm formation. Helvolic acid (2) was known as a fusidane-type antibacterial agent especially against Gram positive bacteria (e.g. Staphylococcus aureus, Enterococus faecalis) as well as a few stains of Gram negative bacteria (e.g. Ralstonia solanacearum and Xanthomonas campestris pv. Vesicatoria) (Sanmanoch et al. 2016;Lv et al. 2017). The current study indicated helvolic acid may be an effective antibacterial agent against S. maltophilia.

General experimental
Optical rotation was measured on an Autopol VI automatic polarimeter. UV and ECD spectra were detected on a Chirascan-plus CD spectrometer. IR spectrum was recorded by the FT-IR-650 spectrometer as KBr disk. 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. High resolution electrospray ionization mass spectrum (HR ESIMS) was recorded on a Shimadzu LC MS-IT-TOF mass spectrometer. Semipreparative HPLC isolation was performed on a LC 3050 N system equipped with UV detector and an Innoval ODS-2 column (10 mm Â 250 mm, 5 lm, 3 mL/min). Silica gel (200-300 and 300-400 mesh) for column chromatography and silica gel GF 254 (10-40 lm) for TLC and preparative TLC were purchased from Qingdao Haiyang Chemical Co. Ltd., China. All solvents were of analytical grade.

Fungal material
Aspergillus udagawae MST1-10 was separated from the soil collected in Xingren coal areas of Guizhou province in China, in May 2020, and identified by ITS method (see Supplementary material S1). This fungus was deposited at the Microbiological Collection Center of Guizhou Medical University (No. GMU-2020-MST 1-10).

Antibacterial assay
The in vitro antibacterial activity was evaluated according to previously reported procedure in 96-well plates (Liu et al. 2020). Nine stains of Gram-negative bacteria (A. baumannii ATCC 19606, A. hydrophila ATCC 7966, E. coli ATCC 25922, K. pneumonia CGMCC 1.839, P. aeruginosa ATCC9027, S. maltophilia ATCC 13637, V. vulnificus NH 87-17, V. alginolyticus ATCC17749, and V. parahaemolyticus ATCC 17802) were prepared with a concentration of 5 Â 10 5 CFU/mL for antibacterial assay. Compounds 1 À 10 were made in 96-well plates with two-fold serial dilution method from starting concentration of 128 lg/mL to the end concentration of 0.5 lg/mL(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. Wells with trimethoprim was served as positive control. 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 2) were successively added into MH culture solution and incubated at 30 C, and the growth was tested by measuring OD 590nm using microplate reader at intervals from 0 to 24 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.

Biofilm inhibition assay
The effect of biofilm inhibition was analyzed by crystal violet staining method (Kannappan et al. 2019;Mashamba et al. 2022). Briefly, A 5 Â 10 5 CFU/mL of S. maltophilia was added to sample groups (with final concentrations of 0.5 MIC, 1 Â MIC, 2 Â MIC and 4 Â MIC), solvent group, and natural group in 96-well plates and then placed in incubator at 30 C for 24 h. The medium was abandoned and washed twice in each well with sterile water, then 95% ethanol was added and fixed for 30 min, and 0.1% crystal violet was stained for 10 min after ethanol sucking out. The crystal violet solution was subsequently removed and washed twice with sterile water. Finally, a Cytation 5 was utilized to capture images at different magnifications as mentioned above. Then, 30% glacial acetic acid was utilized to dissolve the stain, and biofilms were quantified at OD 590nm , and the inhibition rate of biofilm was calculated. Three experiments were conducted in parallel.

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