Marine Mammal Microbiota Yields Novel Antibiotic with Potent Activity Against Clostridium difficile

Abstract

The recent explosion of research on the microbiota has highlighted the important interplay between commensal microorganisms and the health of their cognate hosts. Metabolites isolated from commensal bacteria have been demonstrated to possess a range of antimicrobial activities, and it is widely believed that some of these metabolites modulate host behavior, affecting predisposition to disease and pathogen invasion. Our access to the local marine mammal stranding network and previous successes in mining the fish microbiota poised us to test the hypothesis that the marine mammal microbiota is a novel source of commensal bacteria-produced bioactive metabolites. Examination of intestinal contents from five marine mammals led to the identification of a Micromonospora strain with potent and selective activity against a panel of Gram-positive pathogens and no discernible human cytotoxicity. Compound isolation afforded a new complex glycosylated polyketide, phocoenamicin, with potent activity against the intestinal pathogen Clostridium difficile, an organism challenging to treat in hospital settings. Use of our activity-profiling platform, BioMAP, clustered this metabolite with other known ionophore antibiotics. Fluorescence imaging and flow cytometry confirmed that phocoenamicin is capable of shifting membrane potential without damaging membrane integrity. Thus, exploration of gut microbiota in hosts from diverse environments can serve as a powerful strategy for the discovery of novel antibiotics against human pathogens.

Keywords: cytological profiling; drug discovery; microbiome; natural products; porpoise.

Fig. 1.. Schematic of project workflow.

Isolates collected from marine mammal intestinal contents were fermented, and fractionated into 384 well plates for bioassays. Phocoenamicin was extracted from organism MMA 6B HVS/10A and analyzed as a pure compound.

Fig. 2.. Crude extract BioMAP dilution profiles illustrate potent activity against Gram-positive bacteria.

Natural product fractions on y-axis. Numbering denotes organism extract, RLFI denotes origin of organism, and letters A-G denote polarity of solvent used for extraction, red represents complete growth inhibition, yellow represents growth defects, white represents no growth inhibition.

Fig. 3.. Elucidation of phocoenamicin structure.

(A) Key NMR correlations used in the planar structure elucidation. (B) Newman projection along C₂₁ (front) and C₃₁ and key coupling constant values to determine bond angle. (C) Newman projection along C₃₁ (front) and C₃₂ and key coupling constant values. (D) Newman projection along C₃₂ and C₃₃ and NOE correlations.

Fig. 4.. Chemical Structures of phocoenamicin, relevant spirotetronates and fidaxomicin.

Fig. 5.. Cytological profiling reveals clustering of phocoenamicin with ionophores.

(A) Hierarchical clustering of 5,000 compound treated wells evaluated against 248 cellular features. Positive and negative deviations relative to DMSO controls represented in yellow and blue, respectively. (B) Fluorescence images of drug-treated wells (phocoenamicin 146 μM, monensin 9.6 μM). Tubulin is represented in green, mitotic cells in cyan, actin in red, and DNA in blue. (C) Expanded fingerprints of phocoenamicin and neighboring cluster, which is enriched for ionophores.