Linezolid

Ampicillin sodium (catalogue
no. HY-B0522A, purity: ≥98.0%), vancomycin (catalogue no. HY-B0671,
purity: 98.11%), linezolid (catalogue no. HY-10394, purity: 99.95%),
pinaverium bromide (catalogue no. HY-111613, purity: 99.83%), fluorescent
dye DIBAC4(3) (catalogue no. HY-101892, purity: ≥98.0%), and
chemicals screening libraries (ID: HY-LD-000001025, containing 1333
chemicals, now as part of the FDA-Approved Drug Library HY-L022) were
purchased from MedChemExpress (MCE, Shanghai, China). Phosphatidyl
choline (catalogue no. L130331, purity: >99.0%) and phosphatidyl glycerol (catalogue no. L130372, purity: >99.0%) were purchased from Aladdin (Aladdin, Shanghai,
China). Cardiolipin (catalogue no. C0563, purity: ≥97.0%) and
phosphatidyl ethanolamine (catalogue no. Y0001953, purity: European
Pharmacopoeia Reference Standards) were purchased from Sigma-Aldrich
(Sigma-Aldrich, Shanghai, China).

AF and antibiotics [fosfomycin (FOF); ciprofloxacin (CIP); tetracycline (TET); linezolid (LZD); chloramphenicol (CHL); levofloxacin (LVX); teicoplanin (TEC); clindamycin hydrochloride (CLI) hydro; gentamicin (GEN); vancomycin (VAN)] were purchased from the MedChemExpress company (Monmouth Junction, NJ, United States). E. faecalis ATCC 29212, S. aureus ATCC25923 and ATCC29213 were kindly provided by Juncai Luo (Tiandiren Biotech, Changsha, China). S. epidermidis RP62A and ATCC 12228 were given by Di Qu (Shanghai Medical College of Fudan University), S. aureus ATCC43300 (MRSA), Newman, and RJ-2 were given by Min Li (Renji Hospital, Shanghai Jiao Tong University School of Medicine). Other clinical strains were isolated from the wound secretion or sputum of inpatients at the Third Xiangya Hospital of Central South University. Staphylococcus spp. were grown in tryptic soy broth (TSB) broth medium (Solarbio, Shanghai, China), and E. faecalis was grown in brain heart infusion (BHI) broth medium (Solarbio, Shanghai, China) at 37°C.

Ampicillin sodium (catalog no. HY-B0522A), vancomycin hydrochloride (catalog no. HY-17362), linezolid (catalog no. HY-10394), daptomycin (catalog no. HY-B0108), rifampin (catalog no. HY-B0272), minocycline hydrochloride (catalog no. HY-17412), fosfomycin (catalog no. HY-B1075), and licochalcone A (catalog no. HY-N0372) were purchased from MedChemExpress (Shanghai, China). Gentamicin (catalog no. E003632) and glucose-6-phosphate (catalog no. V900924) were purchased from SIGMA-ALDRICH (Shanghai, China).

S. aureus SA113, Mu50, and NCTC 8325 strains were purchased from American type culture collection (ATCC). The 25 clinical isolates of S. aureus, including 15 MSSA isolates and 10 MRSA isolates, 2 E. faecalis isolates, and one isolate of S. agalactiae, A. baumannii, K. pneumoniae isolate, and E. coli, respectively, were collected from Shenzhen Nanshan People's Hospital and used in this study. The species identification of these bacteria isolates was performed by MALDI-TOF mass spectrometry (IVD MALDI Biotyper, Bruker, Karlsruhe, Germany). S. aureus, E. faecalis, and S. agalactiae strains were grown in tryptic soy broth (TSB) (OXOID, Basingstoke, UK) at 37°C. A. baumannii, K. pneumoniae, and E. coli strains were grown in Luria broth (LB) (1% tryptone, 0.5% NaCl, and 0.5% yeast extract) at 37°C. Oxacillin (catalog no. HY-B0465), vancomycin (catalog no. HY-B0671), linezolid (catalog no. HY-10394), and clemastine (catalog no. HY-B0298A) were purchased from MedChem-Express (MCE, Shanghai, People’s Republic of China). The dimethylsulfoxide (DMSO) was used to dissolve clemastine and equal volumes of DMSO were used as vehicle control in this study. The highest DMSO concentration in the incubation medium was 0.5%, which did not affect bacterial growth and biofilm formation (38 (link)).

The oxazolidinone antibiotics (linezolid, radezolid, and contezolid) and other antibiotics used in this experiment were purchased from MedChemExpress (MCE, Shanghai, China). The minimum inhibitory concentration (MIC) of these antimicrobial agents was determined using the agar dilution method based on Clinical and Laboratory Standards Institute (CLSI) guidelines as described in our previous study (Zheng J. et al., 2020 (link)). While no standard breakpoint was recommended for radezolid against S. aureus in CLSI, in order to analyze the MIC distribution of radezolid, the MICs of radezolid against S. aureus were categorized into three levels, namely, ≤0.125, 0.25, and 0.5 mg/L, and those of linezolid were divided into ≤0.5, 1, 2, and 4 mg/L.

All commercial chemicals and reagents were solubilized and stored according to the manufacturer’s recommendations. Ampicillin sodium salt, d-cycloserine, erythromycin, ethambutol, ethidium bromide, ethionamide, fluorescein sodium salt, gentamycin, kanamycin sulfate, penicillin G sodium, polymyxin B, resazurin sodium salt, RIF, nisin from Lactococcus lactis, tetracycline, and vancomycin were all purchased from Sigma-Aldrich. Bedaquiline, linezolid, macozinone, pretomanid, spectinomycin, streptomycin sulfate salt, and SQ109 were all purchased from MedChem Express. Anhydrotetracycline was purchased from Thermo Fisher Scientific, hygromycin from Roche, and fusidic acid sodium salt from Merck. Compounds from the benzothiazole-core library were stored as stock solutions (10 mM) in DMSO at −20°C.