*3.6. Antibacterial and Antifungal Assays*

Fifty bacterial strains out of 137 were selected on the basis of color and morphological appearance, from which 24 were sequenced and their respective 16S rDNA sequences were identified by BLAST (BLASTN 2.8.1+) [46]. Cultures were maintained in marine broth, and pre-cultures were grown on medium 5294 [47] in a rotatory shaker incubator (50 mL, 30 ◦C, XAD resin, 3 days). Precultures were in duplicate. Cultures of the bacteria for secondary metabolite production were grown in Medium 5294 (2% XAD resin, 7 days, 37 ◦C). The XAD resin was used to sequester secondary metabolites from the media. Specifically, the resin was initially extracted with acetone prior to extraction with methanol. The acetone extracts, being more active than the extracts of methanol, were reconstituted in methanol. Aliquots of the acetone extracts in methanol were used for bioassays against target microbial screens and mass spectrometry profiling. Mass spectrometry profiling is invaluable in this context as it provides all the available masses of the extract.

The antibacterial and antifungal assays adopted the method of Jansen and coworkers [40], briefly described below. Aliquots of the extracts, reference antibiotics, and micrococcin P1 (**1**) (1 mg/mL, 20 μL) were tested against Gram-negative *Escherichia coli* strains JW0451-2 and BW25113, respectively, and Gram-positive *Bacillus subtilis* DSM 10 and *Micrococcus luteus* 1790. Antifungal screens utilized *Pichia anomala* and *Mucor hiemalis*. Streptomycin (SIGMA-ALDRICH) was the reference antibiotic for bacteria and nystatin (SIGMA-ALDRICH) was a reference against yeast and fungi, respectively. *Acinetobacter baumannii*, *Citrobacter freundii*, *S.aureus* Newman, *Pseudomonas aeruginosa* PA14, *Mycobacterium smegmatis*, and the yeast *Candida albicans* were used for assays against pathogens. The % scale activity values were determined in 96-well microtiter plates by 1:1 serial dilution in EBS medium (0.5% casein peptone, 0.5% protease peptone, 0.1% meat extract, 0.1% yeast extract, pH 7.0) for bacteria and MYC medium (1.0% glucose, 1.0% phytone peptone, 50 mM HEPES [11.9 g/L], pH 7.0) for yeasts and fungi. The test organisms were cultivated at 30 ◦C and 160 rpm for 24−48 h, and the cell concentration was adjusted to OD600 0.01 for bacteria and OD548 0.1 for yeast and fungi before the test. The antibacterial activities were ranked to % scale (Table 2). The activities of micrococcin P1 (**1**) against *S. aureus* were given an activity score relative to their absorbances and matched with their respective concentrations. The lowest concentration of micrococcin P1 inhibiting the growth of *Staphylococcus aureus* was considered to be the MIC. The IC50 of micrococcin P1 against *S. aureus* used the method in Okanya et al. [41]. An IC50 calculator was used to generate the IC50 regression curve and to obtain the IC50 of micrococcin P1 (**1**) against *S. aureus*.
