Prodigiosin, Violacein, and Volatile Organic Compounds Produced by Widespread Cutaneous Bacteria of Amphibians Can Inhibit Two Batrachochytrium Fungal Pathogens

Abstract

Symbiotic bacteria can produce secondary metabolites and volatile compounds that contribute to amphibian skin defense. Some of these symbionts have been used as probiotics to treat or prevent the emerging disease chytridiomycosis. We examined 20 amphibian cutaneous bacteria for the production of prodigiosin or violacein, brightly colored defense compounds that pigment the bacteria and have characteristic spectroscopic properties making them readily detectable, and evaluated the antifungal activity of these compounds. We detected violacein from all six isolates of Janthinobacterium lividum on frogs from the USA, Switzerland, and on captive frogs originally from Panama. We detected prodigiosin from five isolates of Serratia plymuthica or S. marcescens, but not from four isolates of S. fonticola or S. liquefaciens. All J. lividum isolates produced violacein when visibly purple, while prodigiosin was only detected on visibly red Serratia isolates. When applied to cultures of chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), prodigiosin caused significant growth inhibition, with minimal inhibitory concentrations (MIC) of 10 and 50 μM, respectively. Violacein showed a MIC of 15 μM against both fungi and was slightly more active against Bsal than Bd at lower concentrations. Although neither violacein nor prodigiosin showed aerosol activity and is not considered a volatile organic compound (VOC), J. lividum and several Serratia isolates did produce antifungal VOCs. White Serratia isolates with undetectable prodigiosin levels could still inhibit Bd growth indicating additional antifungal compounds in their chemical arsenals. Similarly, J. lividum can produce antifungal compounds such as indole-3-carboxaldehyde in addition to violacein, and isolates are not always purple, or turn purple under certain growth conditions. When Serratia isolates were grown in the presence of cell-free supernatant (CFS) from the fungi, CFS from Bd inhibited growth of the prodigiosin-producing isolates, perhaps indicative of an evolutionary arms race; Bsal CFS did not inhibit bacterial growth. In contrast, growth of one J. lividum isolate was facilitated by CFS from both fungi. Isolates that grow and continue to produce antifungal compounds in the presence of pathogens may represent promising probiotics for amphibians infected or at risk of chytridiomycosis. In a global analysis, 89% of tested Serratia isolates and 82% of J. lividum isolates were capable of inhibiting Bd and these have been reported from anurans and caudates from five continents, indicating their widespread distribution and potential for host benefit.

Microbiota and skin defense peptides may facilitate coexistence of two sympatric Andean frog species with a lethal pathogen

“…Our results, along with other studies, suggest that the range of microbial species with antifungal potential is wider than previously considered [3]. Since various studies have found that some bacterial morphotypes from specific groups (e.g., Pseudomonas, Serratia) are effective inhibitors of the fungal pathogen [27,60], further research is necessary to determine if these new candidates meet the requirements to be used as probiotics. It is also key to determine the inhibition spectrum of symbiotic bacteria because antifungal capacities may differ among Bd strains [61,62].…”

The Skin Microbiome of the Neotropical Frog Craugastor fitzingeri: Inferring Potential Bacterial-Host-Pathogen Interactions From Metagenomic Data

“…In amphibians, some members of the skin microbiota can produce secondary metabolites that inhibit the growth of the fungal pathogen Bd. Examples of these antifungal metabolites are violacein (Brucker et al, 2008b ), prodigiosin (Woodhams et al, 2017 ), tryptophol (Loudon et al, 2014b ), indole-3-carboxaldehyde (I3C) (Brucker et al, 2008b ) and 2,4-diacetylphloroglucinol (2,4 DAPG) (Brucker et al, 2008a ).…”

Population dynamics ofRana sierraeat Dusy Basin: influence of non‐native predators, drought, and restoration potential

“…Understanding the mechanisms by which some populations resist Bd could inform management strategies for threatened amphibians. can produce antifungal compounds that reduce Bd load (Brucker et al 2008, Woodhams et al 2018. Whether individual frogs survive or succumb to disease may be in part a result of the rate of their microbiome response to Bd proliferation (Jani and Briggs 2014).…”

“…Strains common to amphibian skin such as Janthinobacterium lividum, Serratia spp., and Pseudomonas spp. can produce antifungal compounds that reduce Bd load (Brucker et al 2008, Woodhams et al 2018). Transplantation of J. lividum from survivors to susceptible individuals has been shown to be most effective in protecting against new Bd outbreaks when applied to the tadpole stage (Jin Song et al 2019).…”