Skin-associated lactic acid bacteria from North American bullfrogs as potential control agents of Batrachochytrium dendrobatidis

NIEDERLE, MARIA V.; J. BOSCH; CESAR EMMANUEL ALE; MARIA ELENA FÁTIMA NADER; CECILIA ARISTIMUÑO FICOSECO; SERGIO PASTERIS,

PLOS ONE

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2020 p. 1 - 23

1932-6203

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosisand has been a key driver in the catastrophic decline of amphibians globally.While many strategies have been proposed to mitigate Bd outbreaks, few have been successful.In recent years, the use of probiotic formulations that protect an amphibian host bykilling or inhibiting Bd have shown promise as an effective chytridiomycosis control strategy.The North American bullfrog (Lithobates catesbeianus) is a common carrier of Bd and harboursa diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial groupcontaining species classified as safe and conferring host benefits. We investigated beneficial/probiotic properties: anti-Bd activity, and adhesion and colonisation characteristics(hydrophobicity, biofilm formation and exopolysaccharide-EPS production) in two confirmedLAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challengedLABs against eight genetically diverse Bd isolates and found that 32% of the LABsinhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a scoreof sensitivity from highest (BdGPL AVS7) to lowest (BdGPL C2A) for the studied Bd isolates.We further reveal key factors underlying host adhesion and colonisation of LABs. Specifically,90.3% of LABs exhibited hydrophilic properties that may promote adhesion to thecutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with asurface polarity compatible with colonisation of acidic, basic or both substrate types. Wealso found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at differentlevels: 21% weak, 29% moderate, and 16.1% strong. Together all these properties enhance colonisation of the host surface (mucus or epithelial cells) and may confer protectivebenefits against Bd through competitive exclusion. Correspondence analysis indicatedthat biofilm synthesis was LABs specific with high aggregating bacteria correlating withstrong biofilm producers, and EPS producers being correlated to negative biofilm producingLABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrateda higher degree of genetic diversity among rod-shaped pLAB than cocci. Basedon the LAB genetic analysis and specific probiotic selection criteria that involve beneficialproperties, we sequenced 16 pLAB which were identified as Pediococcus pentosaceus,Enterococcus thailandicus, Lactobacillus pentosus/L. plantarum, L. brevis, and L. curvatus.Compatibility assays performed with cLAB and the 16 species described above indicate thatall tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggestthat E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18Brepresent optimal probiotic candidates for Bd control and mitigation.