*4.2. Fermentation and Metabolite Extraction*

All twenty-five strains were pre-cultured (5 mL, 28 ◦C, 160 rpm for 7 days) in ISP2 medium [82], ISP3 medium [82] A1M1 medium [27] and 10-fold diluted TSB medium (BD™) prepared in distilled water. Instant ocean (18 g/L) was added in each of them. Each culture [ISP2/ISP3/A1M1/10-fold dil. TSB medium (50 mL) with activated HP-20 resin (Sigma) (2.5 g)] was inoculated (5% *v*/*v* pre-culture) and fermented (14 days, 28 ◦C, 160 rpm). The culture was then centrifuged (4000 rpm, 20 min), the supernatant removed, and the cell/resin pellet lyophilised until dry (Thermo Savant MicroModulyo, Thermo Fisher Scientific, Waltham, MA, USA). The lyophilised cell/resin pellet was extracted twice with ethyl acetate (Fisher Scientific, Loughborough, UK, reagent grade) (20 mL, 100 rpm, 25 ◦C). The extracts were combined, dried (under N2), the weight recorded and stored (4 ◦C).

### *4.3. Bioactivity Disc Diffusion Assay*

Cultures in TSB (BD™) were prepared for *Escherichia coli*, *Staphylococcus aureus*, *Klebsiella pneumonia*e, *Acinetobacter baumanii,* and *Pseudomonas aeruginosa*, whereas cultures in LB [peptone 10 g/L, yeast extract 5g/L, sodium chloride 5 g/L] (5 mL, 30 ◦C, 1200 rpm, 12 h) were prepared for *Enterococcus faecalis*. Nutrient agar (NA, 5 mL, ThermoFisher Scientific) was inoculated with 0.1 mg/mL of the pathogen and was poured onto NA Petri plates (10 mL). The ethyl acetate metabolite extracts were re-dissolved in ethyl acetate at a concentration of 5 mg/mL and 20 μL was added onto each sterile disc (5 mm). The plates were incubated overnight at 30 ◦C and the zones of inhibition were recorded (cm).

#### *4.4. Bioactivity Agar Plug Assay*

The 25 Polar strains were cultured in ISP2, ISP3, 10-fold diluted TSA and A1M1 media in 6-well plates (38 mm diameter/3 mL of media) until a uniform lawn was formed (25 ◦C, 7–14 days). Cultures in TSB (BD™) and LB [peptone 10 g/L, yeast extract 5g/L, NaCl 5 g/L] (5 mL, 30 ◦C, 1200 rpm, 12 h) were prepared for *Escherichia coli*, *Staphylococcus aureus*, *Klebsiella pneumonia*e, *Acinetobacter baumanii, Pseudomonas aeruginosa* and *Enterococcus faecalis*, respectively. NA (5 mL, Thermo Fisher Scientific) was inoculated with 0.1 mg/mL of the pathogen and poured onto NA (10 mL). Plugs (8 mm) from each bacterial lawn (grown for 14 days) were placed on the seeded pathogen plates and incubated overnight at 30 ◦C and the zones of inhibition were measured (cm).
