Polymyxin b

Antibiotic susceptibility testing was implemented in accordance with the Kirby–Bauer disk diffusion method following the Clinical and Laboratory Standards Institute (CLSI) guidelines [47 (link)]. In brief, 150 µL of overnight Vibrio culture was spread aseptically on LB agar plates. E. coli DH5α was used as the negative control (usually E. coli strain K12 MG1655 is used as a negative control). The antibiotics which are most commonly used for the treatment of cholera were chosen for antibiotic susceptibility testing [15 (link),33 (link)]. Different antibiotics discs [ampicillin (AMP, 10 μg), chloramphenicol (C, 30 μg), cefotaxime (Ce, 30 μg), ciprofloxacin (Cf, 5 μg), co-trimoxazole (Co, 25 μg), polymyxin-B (PB, 300 unit), furazolidone (FR, 100 μg), gentamycin (GEN, 10 μg), neomycin (N, 30 μg), norfloxacin (Nx, 10 μg), nalidixic acid (Na, 30 μg), tetracycline (T, 30 μg), cephalexin (CN, 30 μg), streptomycin (S, 10 μg), trimethoprim (Tr, 5 μg)] (HiMedia, Mumbai, India) were placed at certain distances on the surface of the agar plate. The plates were incubated at 37 °C for 24 h and then the diameter of the zone of inhibition of each antibiotic disc was measured.

Antibiotic susceptibility of bla_NDM-1 harboring parent strains, transformants and transconjugants were determined by Kirby Bauer disc-diffusion method including piperacillin-tazobactam (100/10 μg), co-trimoxazole (25 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), polymyxin B (300units), netilmicin (30 μg), carbenicillin (100 μg), tigecycline(30 μg) and faropenem (5 μg) (Hi-Media, Mumbai, India). MICs of imipenem, meropenem, ertapenem, cefepime, aztreonam, gentamicin, amikacin, ciprofloxacin, piperacillin-tazobactam & polymixin-B were determined for parent strains harboring bla_NDM-1, as well as transformants and transconjugants by agar dilution method. Each stock solution for the corresponding antibiotic was made at 1 mg/ml concentration in nuclease free water and was stored at −80 °C. The quality control for stock solution was checked each time against E. coli ATCC 25922. The result of the susceptibility testing was interpreted as per CLSI guidelines [24 ]. However, for polymyxin B, faropenem and carbenicillin, the organisms were considered as non susceptible if the MIC value was higher and diameter of the zone of inhibition was lower than the values given in CLSI guidelines for respective antibiotics against E. coli ATCC 25922.