Piperacillin

For establishing the MIC, the following antibiotics were used; amikacin, meropenem, levofloxacin, chloramphenicol, gentamycin, tobramycin, ceftriaxone and piperacillin (HiMedia, India). Quercetin (Cat No: Q4951) purchased from Sigma Aldrich (USA) was used.
The MIC of antibiotics and Quercetin against the strains were tested by broth micro-dilution method using 96-well plates, using Muller Hinton broth (MHB) (HiMedia, India) according to Clinical & Laboratory Standards Institute (CLSI) guidelines for MIC breakpoints [18 (link)]. Briefly, the antibiotics stocks (10 mg/mL) were prepared using sterile distilled water and Quercetin stock was prepared in dimethyl sulfoxide (DMSO) (10 mg/mL). Fresh MHB containing antibiotics (amikacin, tobramycin, levofloxacin, gentamycin and ceftriaxone) in a broad range between 0.5 to 128 μg/mL was inoculated with overnight cultures of the bacteria at a cell density of 1×10⁵ CFU/mL and incubated at 37°C for 24 h. The antibiotics showing difference of more than 4 μg/mL between visible growth and no visible growth, were further tested in a narrow range between the two concentrations with an increment of 1 μg/mL to establish the exact MIC value. After incubation, the plates were observed for visible growth and MIC was interpreted as the lowest concentration of the antibiotic at which no visible growth was observed.

The antimicrobial susceptibility of the isolated P. aeruginosa isolates was done by the Kirby-Bauer disc diffusion method^{35 (link)}, according to the CLSI guidelines, 2018³⁶ . The following antimicrobial discs were used (Hi-Media Laboratories Ltd., India): Amikacin (30 µg), Piperacillin (100 µg), Ceftriaxone (30 µg), Ciprofloxacin (5 µg), Cefoperazone-Sulbactam (25 µg), Imipenem (10 µg) and Meropenem (10 µg).