Ampicillin discs

The Kirby–Bauer disc diffusion method [17 ] was used to determine the ARP of bacterial cells. The cells were grown overnight in the presence of half of the minimum inhibitory concentration (MIC) dose of EECRR (192.4, 48.4, and 196.4 µg/mL for PU-CA-14, S. aureus ATCC 25923, and MC-CL-10, respectively, as determined in our previous studies [13 (link),14 (link)]). The turbidity of this overnight culture was adjusted by diluting with MHB to make it equivalent to 0.5 McFarland standard so that the culture contains approximately 1.5 × 10⁸ CFU/mL of bacterial cells. Then, 100 µL of such culture was spread over a sterile nutrient agar plate and allowed to stand for 30 min, and commercially available ampicillin discs (10 µg/disc, HiMedia, India) were placed aseptically at the centre of the plate and were incubated at 37°C for 24 h. For the maintenance of proper testing conditions and interpretation of the result, clinical and laboratory standards institute guideline was followed [18 ].

Mueller–Hinton agar, Mueller–Hinton broth (MHB), and ampicillin discs were purchased from HiMedia (Mumbai, India). Tris–HCl, Tris base, ethylenediaminetetraacetic acid (EDTA), agarose, sucrose, 1 kb DNA ladder, bovine serum albumin (BSA), tris buffered saline (TBS)–Tween-20, and Triton-X 100 were purchased from SRL (India). Sodium chloride, disodium hydrogen phosphate, di-hydrogen sodium phosphate, methanol, ethanol, acetic acid, sodium hydroxide, sodium dodecyl sulphate (SDS), and sodium acetate were purchased from E Merck (India), and water was deionized using Millipore Milli-Q system (Bedford, MA). Acridine orange (AO), PBP2a monoclonal antibody, lysostaphin, and proteinase K were purchased from Sigma-Aldrich (St. Louis, MO, USA). An anti-GAPDH monoclonal antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were purchased from Promega (Madison, WI, USA). Ethylene glycol tetraacetic acid (EGTA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyvinylidene fluoride (PVDF) was purchased from Merck-Millipore (Bengaluru, India).