Kanamycin

Antibacterial susceptibility testing was performed using the disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) documents (13 ). The antibiotics used comprised of piperacillin (100 μg), ceftazidime (30 μg), cefotaxime (30 μg), cefazoline (30 μg), tetracycline (30 μg), kanamycin (30 μg), Imipenem (10 μg), and Meropenem (10 μg) (Himedia, Mombay, India). Minimum inhibitory concentration (MIC) for Imipenem was determined by E-test method (AB Biodisk, Solna, Sweden) for all K. pneumoniae isolates according to CLSI guideline (14 (link)).

The susceptibility/resistance of wild-type strains to antibiotics was investigated using the disk diffusion assay. The antibiotics (HiMedia Laboratories, Mumbai, India) studied were gentamycin (10 μg/disk), kanamycin (30 μg), streptomycin (10 μg), tetracycline (30 μg), erythromycin (15 μg), clindamycin (2 μg), chloramphenicol (30 μg), ampicillin (10 μg), and vancomycin (30 μg). The method involved inoculating 4 mL of sterile agar at 45 °C with 200 μL of each culture separately, followed by coating the mixture on MRS agar Petri dishes. After 15 min, the antibiotic disks were placed on the surface, followed by incubation at 37 °C for 24 h and determination of inhibition bands around the disks [37 (link)]. The results were interpreted according to the cut-off levels proposed by Charteris et al. (1998), with strains considered resistant if inhibition zone diameters were equal to or smaller than 12 mm for gentamycin and ampicillin, 13 mm for kanamycin, erythromycin, and chloramphenicol, 11 mm for streptomycin, 14 mm for tetracycline and vancomycin, and 8 mm for clindamycin [37 (link)].

Antibiotic susceptibility testing was done using disk diffusion method as described by Clinical and Laboratory Standards Institute [35 ]. 95 isolates were randomly selected for convenience from all bacteria isolated from the three counties. The isolates were tested for susceptibility to commonly used antibiotics including Ampicillin, Tetracycline, Streptomycin, Co-trimoxazole, Kanamycin, Gentamicin, Sulphamethoxazole, and Chloramphenicol (HiMedia).
Isolates were grown on blood agar for 24 hours; 5 colonies were picked from each plate and suspended in 5 ml of sterile normal saline which was then adjusted to a density approximately equal to McFarland Opacity Standard number 0.5. A dry sterile cotton swab was then placed inside the suspension; excess liquid from the swab was expressed against the wall of the tube and the swab used to spread the bacterial suspension evenly on the surface of Mueller-Hinton agar in order to get confluent growth. Antibiotic disks were then placed on the surface of the inoculum and incubated for 18–24 hours. Zones of inhibition were measured to the nearest millimeter and interpretation as to whether the bacterium was resistant or susceptible to the particular antibiotic was done according to specifications defined by Clinical and Laboratory Standards Institute (CLSI 2006).

Ouabain (OBN) and ATP (adenosine 5′-triphosphate), FITC (fluorescein isothiocyanate), Crystal violet, used in this study were purchased from the Sigma Aldrich (St. Louis, MO). Antibiotics such as ampicillin (AMP), tetracycline (TET), vancomycin (VAN), gentamycin (GEN), amikacin (AMK) and kanamycin (KAN) was procured from HiMedia (Mumbai, India).
The methicillin-sensitive S. aureus (MSSA); ATCC29213 was used in this study. Clinical S. aureus strains IGMS 002 and IGMS 007 received a kind gift from the Indira Gandhi Institute of Medical Science (IGIMS), Patna, were used for cell susceptibility analysis. Muller Hinton Agar/Broth (MHA/MHB) and Tryptic Soya Broth (TSB) were used to culture S. aureus and biofilm formation, respectively at 37 °C. Culture media was purchased from the Hi-media Laboratory.