Gentamicin

Antibiotic susceptibility tests of all isolates were performed using Kirby Bauer disc diffusion method on Mueller-Hinton Agar with recommended antibiotics by CLSI 2020 guidelines [14 ].The antibiotics used were gentamicin (GEN,30 µg), amikacin (AK, 10 µg), ciprofloxacin (CIP, 5 µg), ceftazidime (CAZ, 30 µg), cefepime (CPM, 30 µg), aztreonam (AT, 30 µg), imipenem (IPM, 10 µg), piperacillin (PI,30 µg), piperacillin-tazobactam (PIT), meropenem (MRP, 10 µg), ofloxacin (OF, 30 µg), Levofloxacin (LEV, 30 µg) and colistin (CL,10 µg) from Hi-Media, Laboratories Pvt. Ltd. India.
Isolates that were non-susceptible to at least one agent in ≥ 3 antimicrobial categories have been categorized under MDR [15 (link)].

The isolates positive for at least one virulence marker gene by PCR were subjected to antimicrobial susceptibility test against the selected antimicrobials (ampicillin-10 μg, amikacin-10 μg, chloramphenicol-30 μg, ceftriaxone-10 μg, cephalexin-30 μg, ciprofloxacin-10 μg, co-trimoxazole-25 μg, cefoperazone-tazobactam-75 + 10 μg, meropenem-10 μg, norfloxacin-10 μg, gentamicin-10 μg, cefixime-5 μg, doxycycline hydrochloride-10 μg and ofloxacin-5 μg) (HiMedia, India) by disc diffusion method in Mueller–Hinton agar [18 (link)]. The performance of this test was checked by employing E. coli ATCC 25922 as a standard quality control strain. The results were expressed as sensitive, intermediate and resistant as per standard CLSI guidelines [19 ]. MDR was defined as “acquired non-susceptibility to at least one agent in three or more antimicrobial categories” [20 (link)].

The antimicrobial susceptibility testing of honey samples against E. coli was determined by the disc diffusion method described by Bauer et al. at the honey concentration of 700 mg/disc [49 (link)]. Briefly, bacterial colonies were isolated from pure culture and inoculated in 5 mL of tryptic soy broth (Merck, Darmstadt, Germany), then incubated at 37 °C for 2–5 h [49 (link)]. The optical density (O.D) was measured spectrophotometrically and adjusted between 0.08 and 0.1 at 600 nm, which was approximately equal to 10⁸ CFU/mL (corresponding to 0.5 McFarland standard) [48 ,50 (link)]. Subsequently, bacterial suspension was spread evenly on an agar plate by a sterile cotton swab. Sterile 6.0 mm diameter blank discs (HiMedia, Mumbai, India) were impregnated with 50 µL of the honey concentration and vehicle as a negative control separately, then placed on the surface of the inoculated plate using sterile forceps. Gentamicin 10 µg/disc (Himedia, Mumbai, India) was used as a positive reference control. The plates were incubated aerobically at 37 °C for 24 h. The inhibition zone diameter was measured in mm, and the average was calculated. The experiment was repeated in triplicates for each isolate.

All E. coli isolates were tested for antibiotics susceptibility by using the disk diffusion method as reported by Kirby-Bauer [18 (link)]. Briefly, isolates were suspended in sterile 0.85% normal saline and adjusted to 0.5 McFarland standard solution. Then, MHA plates were inoculated, and antibiotic disks were seeded within 15 min after inoculation of MHA plates. MHA plates were incubated aerobically at 37 °C for 16–18 h. The interpretations of zones of inhibitions were performed as recommended by the CLSI 29th Edition guidelines [19 ]. All E. coli that showed intermediate susceptibility to the antibiotics tested were regarded as resistant to such particular antibiotics. Antibiotics tested included ciprofloxacin (CIP 5 μg; HiMedia, Mumbai, India), ampicillin (AMP 10 μg; HiMedia, India), tetracycline (TE 30 μg; HiMedia, India), meropenem (MEM 10 μg; HiMedia, India), ceftazidime (CAZ 30 μg; HiMedia, India), gentamicin (CN 10 μg; HiMedia, India), cefepime (FEP 30 μg; HiMedia, India), and trimethoprim-sulfamethoxazole (SXT 25 μg; HiMedia, India).

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.