AUTHOR=Loudon Andrew H. , Holland Jessica A. , Umile Thomas P. , Burzynski Elizabeth A. , Minbiole Kevin P. C. , Harris Reid N. 

TITLE=Interactions between amphibians' symbiotic bacteria cause the production of emergent anti-fungal metabolites

JOURNAL=Frontiers in Microbiology

VOLUME=5

YEAR=2014

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00441

DOI=10.3389/fmicb.2014.00441

ISSN=1664-302X

ABSTRACT=<p>Amphibians possess beneficial skin bacteria that protect against the disease chytridiomycosis by producing secondary metabolites that inhibit the pathogen <italic>Batrachochytrium dendrobatidis</italic> (<italic>Bd</italic>). Metabolite production may be a mechanism of competition between bacterial species that results in host protection as a by-product. We expect that some co-cultures of bacterial species or strains will result in greater <italic>Bd</italic> inhibition than mono-cultures. To test this, we cultured four bacterial isolates (<italic>Bacillus</italic> sp., <italic>Janthinobacterium</italic> sp., <italic>Pseudomonas</italic> sp. and <italic>Chitinophaga arvensicola</italic>) from red-backed salamanders (<italic>Plethodon cinereus</italic>) and cultured isolates both alone and together to collect their cell-free supernatants (CFS). We challenged <italic>Bd</italic> with CFSs from four bacterial species in varying combinations. This resulted in three experimental treatments: (1) CFSs of single isolates; (2) combined CFSs of two isolates; and (3) CFSs from co-cultures. Pair-wise combinations of four bacterial isolates CFSs were assayed against <italic>Bd</italic> and revealed additive <italic>Bd</italic> inhibition in 42.2% of trials, synergistic inhibition in 42.2% and no effect in 16.6% of trials. When bacteria isolates were grown in co-cultures, complete <italic>Bd</italic> inhibition was generally observed, and synergistic inhibition occurred in four out of six trials. A metabolite profile of the most potent co-culture, <italic>Bacillus</italic> sp. and <italic>Chitinophaga arvensicola</italic>, was determined with LC-MS and compared with the profiles of each isolate in mono-culture. Emergent metabolites appearing in the co-culture were inhibitory to <italic>Bd</italic>, and the most potent inhibitor was identified as tryptophol. Thus mono-cultures of bacteria cultured from red-backed salamanders interacted synergistically and additively to inhibit <italic>Bd</italic>, and such bacteria produced emergent metabolites when cultured together, with even greater pathogen inhibition. Knowledge of how bacterial species interact to inhibit <italic>Bd</italic> can be used to select probiotics to provide amphibians with protection against <italic>Bd</italic>.</p>