*2.3. Production of Secondary Metabolites*

A single colony from freshly grown culture plate (72 h) was inoculated in 50 mL SCN broth (pH 7.4), which was incubated at 28 ◦C for 72 h to develop the pre-seed culture. The production medium (SCN broth, pH 7.2) was inoculated with the pre-seed culture (1%; *v*/*v*) to commence production of the secondary metabolites, which was carried out for 7 days at 28 ◦C in an incubator shaker (Adolf Kuhner AG, Birsfelden, Basel, Switzerland) run at 200 rpm. The cell-free broth was recovered by centrifugation at 5000× *g* for 20 min in Sorvall RC 5C plus centrifuge (Kendro Laboratory Products, Newtown, Connecticut, USA). The metabolites were recovered from the supernatant by using liquid-liquid extraction with equal volume of ethyl acetate. The extracted metabolites were dried by using rotary evaporator (50 ◦C) and vacuum oven (35 ◦C). The dried metabolite preparations were stored at 25 ◦C ± 2 ◦C till further use.

### *2.4. Antimicrobial Susceptibility Testing*

The reference strains of bacterial pathogens used in this study were: *S. aureus* ATCC 29213, *S. aureus* ATCC 25923, *S. aureus* ATCC 13709, MRSA ATCC 43300, MRSA 562, *S. epidermis* ATCC 12228, *Enterococcus faecalis* ATCC 29212, *E. faecium* ATCC 49224 and *E. faecium* AIIMS. In-vitro antibacterial activity of the metabolite extract was determined on cation adjusted Muller Hinton agar (MHA) (Himedia, Mumbai, India) plates using well diffusion method [24]. The minimum inhibitory concentration (MIC90) values were measured in a 96-well microtiter plates by the broth microdilution method as per the guidelines of Clinical and Laboratory Standards Institute (CLSI) [33]. Briefly, a stock solution of the metabolite extract (1mg/mL) was prepared in 0.2% DMSO and cation adjusted Muller Hinton broth. Bacterial pathogens (100 <sup>μ</sup>L; 2 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL) and metabolite extract (100 <sup>μ</sup>L) at concentrations varying from 125 to 0.122 μg/mL were added to the individual well in the microtitre

plate. A sample control (ADR1 extract alone) and blank (media only) were included in each assay. After incubation for 24 h at 37 ◦C, iodonitrotetrazolium chloride (INT) (Sisco Research Laboratories, Mumbai, India) was added to the wells and the plates were incubated further for 30 min. The absorbance was measured on a multimode reader (Biotek Instruments, Winooski, Vermont, USA) at 490 nm. The value of MIC90 was considered to be the minimum concentration at which no visible growth could be observed. The following equation was used to compute the percent inhibition [34].

Growth inhibition of pathogen (%) = [(control OD490 nm − test OD490 nm)/control OD490 nm] × 100 (1)
