Beef extract

Glucose, beef extract, bacteriological peptone, biochemical (KB009) and antibacterial (IC002) assay kits were obtained from Hi-Media (India). All HPLC grade organic solvents like methanol, dichloromethane and acetonitrile were procured from Merck (India). Zeaxanthin standard was procured from DHI, Denmark. The bacterium used in this study, Arthrobacter gandavensis, is available in Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology, Chandigarh with the Accession number MTCC 25325.

Media components, Beef Extract (catalog number: RM002), Peptone (catalog number: CR001), Yeast Extract (catalog number: RM027) and Sodium Chloride (catalog number: TC046) for bacterial culture were purchased from HiMedia Laboratories (Mumbai, India). Anti-phosphoserine antibody was purchased from Sigma-Aldrich Chemical Co., St. Louis, MO (catalog number: SAB5200086-400UL). Inhibitor molecules were purchased from Enamine Ltd., Ukraine. PAGE reagents and buffers were purchased from Biorad and PVDF transfer membrane, 0.2 µm (Catalog number: 88520) was purchased from Thermo Fisher Scientific, Inc. Mini-PROTEAN® Tetra Cell, 2-gel, 10-well combs, 1.0 mm (catalog no. 1658003) from Biorad was used for PAGE and blotting was performed using semi-dry blotting unit (Scie-Plas Ltd., UK). Sensitive and resistant E. faecalis (Nis^R-147) bacteria were previously isolated from raw buffalo milk in our lab [5 (link), 23 (link)]. The nisin-resistant strain used here was found to be resistant to chloramphenicol, ampicillin, ciprofloxacin, rifampicin, vancomycin, carbenicillin, linezolid, oxacillin, and fosfomycin with minimum inhibitory concentrations (MICs) varying between 4 µg/mL (ciprofloxacin) and 512 µg/mL (fosfomycin) [5 (link)].

The A. thaliana (Col‐0) plants were grown in a growth chamber (22°C and 50%–60% relative humidity) with a 16/8 h light/dark period. The rice TN1 seeds were soaked in water overnight and germinated on filter paper in a Petri plate. Germinated seedlings were transferred to pots and maintained for 45 days in the greenhouse (28°C and 50%–60% relative humidity) with a 16/8 h light/dark photoperiod. The Xoo strain was cultured in nutrient broth (NB) in a shaking incubator at 28°C for 2 days. Nutrient broth was prepared using peptone 5 g, NaCl 5 g, beef extract 1.5 g and yeast extract 1.5 g (HiMedia) in 1 L of distilled water (pH 7–7.2). Nutrient agar (NA) was prepared using peptone 5 g, NaCl 5 g, beef extract 1.5 g, yeast extract 1.5 g and agar powder 15 g (Sigma) in 1 L of distilled water.

All the new N-substituted 1,2,3-triazolylmethyl indole derivatives were evaluated for antimicrobial activity against various bacterial and fungal cultures at a concentration of 10, 20 (μM) employing the well diffusion method.³⁵ The standard antibiotic streptomycin was employed as standard. The well diffusion method was carried out on nutrient agar medium (0.5% peptone, 0.5% NaCl and 0.3% beef extract, HiMedia, Mumbai). The nutrient agar medium was autoclaved at 121 °C/15 lbs, after the autoclaving medium was cooled (40 °C) and poured into Petri dishes (Borosil, S-Line). After solidification, the Petri dishes were observed for sterility by overnight incubation. After the sterility check, the medium was cultured with 10⁸ cfu mL⁻¹ actively grown (0.1 mL) bacterial culture using the spread plate technique. After a few minutes, wells were created on the cultured medium using a sterile well borer (5 mm) and selected dilutions of the synthesized N-substituted 1,2,3-triazolylmethyl indole derivatives with selected concentrations were inoculated. After inoculation, the cultured plates were incubated at 37 °C/24 h, and the antimicrobial activity was evaluated based on the zone of inhibition (mm) around the well.