Netilmicin

Antimicrobial sensitivity testing was done by Kirby-Bauer Antimicrobial Susceptibility Test (disc diffusion method) using Mueller-Hinton agar [15 ]. Six different antibiotics were tested and the zone size was measured. Amikacin, streptomycin, gentamicin, tobramycin, netilmicin, and neomycin (obtained from HiMedia, India) were taken and screening test for detection of high level aminoglycoside resistance (HLAR) in Enterococcus species was confirmed as per Clinical and Laboratory Standards Institute (CLSI) approved standards [16 ] The E. coli isolates were described as isolates with high level aminoglycoside resistance considering growth ≥2048 μg/mL for streptomycin, ≥512 μg/mL for gentamicin, ≥512 μg/mL for neomycin, ≥256 μg/mL for tobramycin, ≥256 μg/mL for netilmicin, and ≥256 μg/mL for Amikacin. MICs were determined by Etest (AB Biodisk).

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.

The antimicrobial susceptibility was performed by the Kirby Bauer's disc diffusion technique on Mueller-Hinton agar, as per Clinical Laboratory Standard Institute (CLSI) guidelines [5 ]. The antibiotics tested were as follows (potency in μg/disc): Ampicillin (10), Cefuroxime (30), Cefotaxime (30), Piperacillin (100), Ticarcillin (75), Piperacillin-Tazobactam (100/10), Ticarcillin-Clavulanic acid (75/10), Ceftazidime (30), Cefepime (30), Aztreonam (30), Imipenem (10), Meropenem (10), Ertapenem (10), Colistin (10), Gentamicin (10), Tobramycin (10), Amikacin (30), Netilmicin (30), CiprOfloxacin (5), LevOfloxacin (5), Lomefloxacin (10), and Ofloxacin (5) (HiMedia Laboratories Pvt. Ltd., Mumbai, India). P. aeruginosa ATCC 27853, E. coli ATCC 25922, E. coli ATCC 35218, and K. pneumoniae ATCC 700603 were used as quality control strains.