Vibrio cholerae

Lyophilized cultures of Escherichia coli (MTCC 1687); Vibrio cholerae
(MTCC 3906); Proteus mirabilis (MTCC 425); Klebsiella pneumoniae
(MTCC3384); Staphylococcus aureus (MTCC 3160); Salmonella typhi
(MTCC3231) was obtained from Microbial Type Culture Collection,
IMTECH, India. All test strains were cultured at 37 °C in Luria
broth or on Luria agar. Antibacterial activity of cell-free
supernatant was determined using the disc diffusion method. 100
µl of 8 hrs old culture broth of the test organisms were spread over
on the surface of the Luria agar plate using a sterile cotton swab.
After that, 60 µl of cell-free supernatant was added into sterile
standard discs (Himedia, India) and incubated for 18 to 24 hrs at
37 °C. After that the clear incubation zone of inhibition (ZOI) was
measured to evaluate the antimicrobial activity of actinomycetes
isolate. Dimethyl sulphoxide (DMSO) was used as control.

The antibacterial efficacy of the biosynthesised GNP023 was assessed against the bacterial pathogens. Salmonella typhimurium (MTCC 734), Staphylococcus aureus (MTCC737), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA811) and Vibrio cholerae (N16961) were procured from the Microbial Type Culture Collection (MTCC), IMTECH, Chandigarh, India, using the single-disc diffusion method. The test pathogens were seeded in nutrient broth (NB), to reach about 3.0 × 10⁻⁸ CFU/mL overnight. The overnight seeded cultures were, then, transferred to fresh NB medium and grown till 1.5 × 10⁻⁸ CFU/mL (0.5 McFarland scale), and 100 μL of this culture was spread over Muller Hinton Agar (MHA) plates. Sterilised 6 mm Whatman paper No. 1 discs were, aseptically, placed on MHA plates with test cultures. Five microliters (5 µL) of GNP023, prepared in 10% DMSO at different concentrations (400, 200 and 100 µg/disc), as well as AREF023 methanol extract (ME) at concentrations (800, 600 and 400 µg/disc), were loaded, aseptically. Standard antibiotics Vancomycin (S. aureus and MRSA), Chloarmphenicol (S. typhimurium and V. cholera) and 10% DMSO served as controls. The plates were incubated at 37 °C for 24 h and observed for a clear zone of inhibition (in mm). The antibacterial assay was performed in triplicate, and the quantitative variables were represented in terms of mean ± SD [25 ].