Development of a liquid-medium assay for screening antimicrobial natural products against marine bacteria

The marine environment is known to be a promising source of new compounds with biological activities. Many papers report on the discovery of such compounds in marine micro-organisms, especially marine eukaryotes such as micro-algae or fungi. In this context, we describe the development of antibacterial assays in a liquid-medium, to detect an activity within the lipophilic fraction of micro-organisms. Bacteria used for this assay were Vibrio species. Bacterial growth was carried out in a Bioscreen C, and the first step in this development was to determine the tolerance of the Vibrio species to solvents miscible with aqueous culture media. Methanol, ethanol and DMSO were tested and the best response was obtained with methanol, at a concentration of 1.5%. Commonly known antibiotics were then used to calibrate the assay. A preliminary antibacterial assay was carried out with extracts of Azadinium spinosum, a small toxic dinoflagellate, and a marine-derived strain of the fungus Penicillium expansum. A. spinosum showed no activity on V. crassostreae at a concentration of 26.7 mg/mL, whereas a crude extract at 5 mg/mL of P. expansum resulted in an inhibition of V. crassostreae growth. Therefore this assay seems to be able to detect an activity in crude extracts. However, as some crude extracts did not dissolve completely, emulsifying agents may improve the scope of the assay to include more lipophilic compounds.