P110: Rapid detection of Gram-negative antibiotics from sediment-derived marine bacteria

There is an urgent need to find new antibiotics to treat Gram-negative bacterial infections.  In this study, we used Escherichia coli in a soft agar overlay assay to rapidly identify marine bacteria capable of inhibiting Gram-negative bacteria.  Approximately 300 marine sediment samples collected from Fiji, the Channel Islands, the Dry Tortugas, and other locations were plated onto seawater based agar media and incubated until bacterial colonies were formed.  These primary isolation plates were overlaid with E. coli in soft agar and following 24-48 h, colonies showing zones of inhibition were removed and purified using standard microbiological methods.  Thirteen strains were confirmed as inhibiting the growth of E.coli in secondary assays following purification.  It was determined that pH and iron limitation were not the cause of inhibition.  The agar blocks cut from the zones of inhibition demonstrated activity indicating that the active substance(s) were diffusible.  All 13 strains were identified as Bacillus species based on16S rRNA gene sequencing.  Crude butanol, methanol, and acetone extracts generated from the zones of inhibition were all active at a concentration of 10 mg/mL.  The results of the bioassay-guided isolation of the active constituents and correlations between 16S sequence type, structure diversity, and biological activity will be presented.