Mueller hinton agar (mha)

The susceptibility of the reference, clinical and environmental bacterial strains to the crude extract (10.00 mg/mL) obtained from the B. amyloliquefaciens ST34 strain was determined using the method outlined by Ndlovu et al. [27 ]. Briefly, the lyophilised ST34 crude extract was dissolved in 15% (v/v) methanol to obtain a concentration of 10.00 mg/mL. Antimicrobial discs (6 mm; Oxoid, USA) were placed on the MHA after the test isolates were spread plated onto the media and were impregnated with 50 μL of the surfactin crude extract (with approximately 9.98 μg surfactin in the crude extract). For each assay, a surfactin negative and positive control was included in the experiment for each of the test organisms. The negative control consisted of the test microorganism spread plated onto MHA (Merck) with 15% (v/v) methanol impregnated filter paper discs, while the positive control consisted of the reference test organisms [AB 1, EC 1, KP 1 and PA 1] spread plated onto MHA with commercial surfactin (1.00 mg/mL) (Sigma, USA) impregnated filter paper discs added on top of the media. All the agar plates were incubated for 24–48 h at 37 °C, whereafter the diameters of the zones of inhibition around the inoculated paper discs were measured and recorded.

The Kirby–Bauer disk diffusion method was used to evaluate antibiotic resistance in E. coli. This method used Mueller–Hinton agar (MHA, Merck) with the pour method to determine the diameter of antibiotic resistance. This method produces qualitative categories with sensitive, intermediate, and resistant assessments based on the standard Clinical and Laboratory Standards Institute (CLSI) (Table-1) [19 ].
A 0.2 mL suspension of bacteria was put in a Petri dish containing MHA (Merck) and, then leveled with a bent glass rod so that the bacteria stick to the medium. The bacteria were allowed to stand for 10–15 min. The antibiotic (Oxoid, UK) on the disk was placed on the surface, and then the plate was incubated at 37°C for 24 h. The diameter of the resulting inhibition zone was measured with a caliper and then compared with the standard from CLSI.

The bacterial strain Aeromonas hydrophila (7966) was purchased from the American Type Culture Collection, Tamil Nadu, India. Nutrient broth, Mueller-Hinton agar, DPPH, and media were purchased from Sigma-Aldrich, India, and the standard antibiotics were purchased from Hi-Media Laboratories, Mumbai, India. In addition, zebrafish (Danio rerio) embryos were purchased from Tarun fish farm, Manimangalam, Chennai.

All the chemicals, microbiological media and reagents, such as, Mueller–Hinton agar, ceftriaxone sodium and tetra chloroauric [III] acid (HAHuCl₄) were procured from Sigma–Aldrich (St. Louis, MO, USA).

Microorganisms used in this study included 36 multidrug-resistant strains of Gram-negative belonging to Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa species and Gram-positive bacteria belonging to and Staphylococcus aureus species (Table 3). Their bacterial features were previous reported [6 (link)]. Mueller–Hinton Agar (Sigma) was used to activate the microorganisms whilst Mueller Hinton broth (MHB; Sigma) was used for antibacterial assays.

2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), potassium persulfate (K₂S₂O₈), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H₂O₂), 2, 4, 6-tripyridyl-s-triazine (TPTZ), gallic acid, quercetin, butylated hydroxyl toluene (BHT), ascorbic acid, Mueller–Hinton agar, and broth were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). All other chemicals were of the analytical grade and were purchased from Merck Limited (Mumbai, India).