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

Abstract

Amphibians possess beneficial skin bacteria that protect against the disease chytridiomycosis by producing secondary metabolites that inhibit the pathogen Batrachochytrium dendrobatidis (Bd). 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 Bd inhibition than mono-cultures. To test this, we cultured four bacterial isolates (Bacillus sp., Janthinobacterium sp., Pseudomonas sp. and Chitinophaga arvensicola) from red-backed salamanders (Plethodon cinereus) and cultured isolates both alone and together to collect their cell-free supernatants (CFS). We challenged Bd 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 Bd and revealed additive Bd 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 Bd inhibition was generally observed, and synergistic inhibition occurred in four out of six trials. A metabolite profile of the most potent co-culture, Bacillus sp. and Chitinophaga arvensicola, 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 Bd, 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 Bd, and such bacteria produced emergent metabolites when cultured together, with even greater pathogen inhibition. Knowledge of how bacterial species interact to inhibit Bd can be used to select probiotics to provide amphibians with protection against Bd.

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

“…In addition, the relative abundance of these OTUs may have increased because Bd was able to negatively affect the abundance of other OTUs [57]. Facilitation of Bd growth and colonization could occur either when symbiotic bacteria shift their metabolite profiles in a way that favours Bd [35] or by reducing the capability of other microbes to inhibit Bd. In a recent study, bacteria isolated from cyclamen and tomato plants, including a Novosphingobium sp.…”

“…Metabolites produced by amphibian cutaneous bacteria can inhibit Bd in vitro [13,34,35] and in vivo [11] . Therefore, we excised and extracted the skin of each frog to obtain cutaneous metabolites following procedures described by Brucker et al [13]; briefly, excised skins were extracted three times by shaking in HPLC-grade methanol (5 ml each extraction).…”

Amphibian Symbiotic Bacteria Do Not Show a Universal Ability To Inhibit Growth of the Global Panzootic Lineage of Batrachochytrium dendrobatidis

“…Studies have repeatedly illustrated the importance in a complex microbiome for resilience of the community in response to a pathogenic infection (33)(34)(35). A bacterial consortium approach that treats microbiomes as a suite of functional traits rather than a substrate for the insertion of candidate bacteria is likely to offer a more comprehensive protection of hosts from B. dendrobatidis and other threatening amphibian pathogens (12,28,36). How the different members of such consortia will be determined is currently unknown, but our results highlight the limitations of a taxonomic approach for understanding what bacterial communities may afford resistance to B. dendrobatidis: both species and genus showed a limited potential to identify potentially inhibitory bacteria in our study.…”

Probiotics as a tool for disease mitigation in wildlife: insights from food production and medicine

“…Multistrain mixtures have also been considered, since a wider variety of antipathogen secretions are expected. In addition, different strains in competition can produce antipathogenic secretions, which are not produced through growth alone . A study in mice found that a mixture of six bacteria cured C .…”