Cefotaxime

Antimicrobial susceptibility of bla_CTX-M harbouring parent strains as well as transformants were determined by Kirby Bauer disc diffusion method and results were interpreted as per CLSI guidelines [17 ]. Following antibiotics were tested: cefotaxime (30μg), cefoxitin (30μg), ceftazidime (30μg), amikacin (30μg), gentamicin (10μg), kanamicin (30μg), ciprofloxacin (5μg), trimithoprim/sulphamethoxazole (1.25/23.75μg), imipenem (10μg), ertapenem (10μg), tigecycline (15μg) and polymyxin B (300 units) (Hi-Media, Mumbai). MIC was also determined for donor strain and transformants against cefotaxime, ceftazidime and ceftriaxone (Hi-Media, Mumbai, India) by agar dilution method.

Transformation in sesame explants were carried using transformed Agrobacterium tumefaciens single colony having the desired gene combination in the binary pBI121 vector. The antibiotics used in selection were kanamycin and rifampicin and the growth conditions was 200 rpm at 28 °C for 12 h. A.tumefaciens liquid culture was centrifuged at 4000× g for 10 min to pellet the cells. The cells were suspended in 50 ml of liquid MS medium and 20 µl of 20 μM acetosyringone (Murashige and Skoog 1962). The de-embryonated cotyledons were incubated in the liquid agrobacterium for 15 min and dried on sterile filter paper. Infected explants were then incubated in dark for 3 days on co-cultivation medium (pre regeneration medium + 20 μM acetosyringone). Following co-cultivation, explants were washed two times with sterile water supplemented with 500 mg/l cefotaxime (Himedia, India) and placed in pre culture media containing cefotaxime (500 mg/l) and 50 mg/l kanamycin. Explants were sub cultured with two week intervals and transferred to shoot elongation medium containing MS basal medium with 0.3 mg/l GA₃.

The pre-enriched samples were streaked onto MacConkey agar (Sigma-Aldrich) supplemented with cefotaxime and ceftazidime (HiMedia, India) at the concentration of 2 μg/mL each and incubated at 37°C for 18-24 h. One colony from each type was picked from primary growth and subcultured on the MacConkey agar plate supplemented with both cefotaxime and ceftazidime at the final concentration of 1 μg/mL. Purified isolates were identified using the procedures described by Cowan and Steel [26 ]. Biochemically confirmed isolates were stored in trypticase soy broth (TSB; Sigma-Aldrich) containing 30% glycerol at −80°C.