Muller hinton broth

All the chemicals and reagents used for this study including; ethanol (96%), methanol (99.8%), petroleum ether (40–60 °C), dichloromethane (≥99.8%), chloroform (99.9%), n-butanol (99.8%), dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), potassium hydroxide (KOH, ≥85%), 1,1-diphenyl2-picrylhydrazyl (DPPH, 95%), 2, 20-azino-bis[3-ethyl benzo thiazoline-6-sulphonic acid] (ABTS), potassium persulfate (K₂S₂O₈), acetylthiocholine iodide (≥99.0%), S-butyrylthiochoilne iodide (≥98.0%), sodium phosphate (Na₃PO₄, 96%), ascorbic acid, resazurin, 2-nitrobenzoic acid and were obtained from Sigma Aldrich (Hamburg, Germany). Muller Hinton agar and Muller Hinton broth were procured from HiMedia (Himedia Laboratories Pvt Ltd., Mumbai, India). Donepezil, galantamine and chloramphenicol were purchased from the local drug store.

Determination of the minimum inhibitory concentration (MIC) was carried out by conventional broth microdilution methods based on the Clinical and Laboratory Standards Institute (CLSI) adapted for antimicrobial peptides [34 (link)]. The overnight cultures of the test strains were diluted in Muller Hinton broth (HiMedia, Mumbai, India) in order to obtain 10⁶ CFU/mL. Prepared inoculums were mixed with two-fold dilutions of emericellipsin A and incubated for 24 h in 96-wells microtiter plate (Eppendorf, Hamburg, Germany). The indolicidin (Research Institute of Highly Pure Biopreparations, Saint Petersburg, Russian Federation), vancomycin (Sigma-Aldrich, St. Louis, MO, USA), and norfloxacin (Sigma-Aldrich) were used as positive controls. After incubation, the optical density of planktonic cells was assessed by reading the absorbance data at 620 nm. These data were obtained by the IEMS MF spectrophotometer (Labsystems, Vantaa, Finland). Antimicrobial activity of emericellipsin A was indicated by the minimal inhibitory concentration, which was defined as the lowest dose at which no visible growth was detected. Determination of bactericidal activity of emericellipsin A was performed by plating of the treated bacteria from the wells on agar medium (Muller Hinton, HiMedia). Following incubation, CFU counting was conducted.

Ethanol (99.80%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Muller-Hinton agar (MHA), Muller-Hinton broth (MHB), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and fetal bovine serum (FBS) were received from HiMedia, Mumbai, India. Calcium nitrate tetrahydrate, orthophosphoric acid, gallic acid, catechin, rutin, Dulbecco’s modified Eagle’s medium (DMEM), antibiotic and antimycotic solution, lipid peroxidation kit, Griess reagent, caspases kits (3/7, 8, and 9), antioxidant enzyme kits (SOD, CAT, and GSH), ELISA kits (TNF-α and IL-6), primer sequences (TNF-α, IL-6, iNOS, COX-2, and β-actin), dichloro-dihydro-fluorescein diacetate (DCFH-DA), TRI reagent, rhodamine 123, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich, Bengaluru, India. The live/dead dual staining kit was purchased from Thermo Fisher Scientific and iScript One-Step RT-PCR kit with SYBR green was obtained from Bio-Rad, Bengaluru, India.

A. baumannii isolates (n = 37) were collected from clinical samples (cerebrospinal fluid) of the patients (n = 37) in the neuro-ICU in the period from January 2013 to September 2020. Bacterial identification was performed using a MALDI-TOF Biotyper (Bruker Daltonics, Bremen, Germany) instrument. Bacterial isolates were grown at 37 °C on Nutrient Medium No. 1 (SRCAMB, Obolensk, Moscow region, Russia), Luria–Bertani broth (Difco Laboratories, Detroit, MI, USA), and Muller–Hinton broth (Himedia, Mumbai, Maharashtra, India). Bacterial isolates were stored in 15% glycerol at minus 80 °C.

Escherichia was cultivated on agar and Muller–Hinton broth (Himedia, Mumbai, India) for 18–20 h at 37 °C. Staphylococcus was cultivated using Nutrientagar and Nutrientbroth (Himedia, India) and the GRM medium (Obolensk, Moscow, Russia) for 18–20 h at 37 °C. Nutrientagar and Nutrientbroth (Himedia, India) were used for Bacillus cultivation, and the culture was grown for 18–24 h. The cultivation of fungi of the genus Candida was conducted on Sabouraud’s medium at 30–37 °C for 48 h.

Nutrient broth, Muller-Hinton broth, and agar powder were purchased from Himedia. Dimethylsulphoxide (DMSO) was purchased from E. Merck. Reference antibiotic disks were purchased from Himedia. The other materials were purchased from E. Merck (India). Compound 10 and compound 11 used in this work were synthesized in our laboratory.