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

PLoS One. 2019 Sep 27;14(9):e0223020. doi: 10.1371/journal.pone.0223020. eCollection 2019.

Abstract

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis and 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 by killing 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 harbours a diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial group containing 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 confirmed LAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60 presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challenged LABs against eight genetically diverse Bd isolates and found that 32% of the LABs inhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a score of 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 the cutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with a surface polarity compatible with colonisation of acidic, basic or both substrate types. We also found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at different levels: 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 protective benefits against Bd through competitive exclusion. Correspondence analysis indicated that biofilm synthesis was LABs specific with high aggregating bacteria correlating with strong biofilm producers, and EPS producers being correlated to negative biofilm producing LABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrated a higher degree of genetic diversity among rod-shaped pLAB than cocci. Based on the LAB genetic analysis and specific probiotic selection criteria that involve beneficial properties, 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 that all tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggest that E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18B represent optimal probiotic candidates for Bd control and mitigation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chytridiomycota / isolation & purification
  • Chytridiomycota / pathogenicity*
  • DNA, Bacterial
  • Lactobacillales / genetics
  • Lactobacillales / immunology*
  • Lactobacillales / isolation & purification
  • Microbiota / immunology*
  • Mycoses / immunology
  • Mycoses / microbiology
  • Mycoses / veterinary*
  • Probiotics / isolation & purification*
  • Rana catesbeiana / immunology
  • Rana catesbeiana / microbiology*
  • Random Amplified Polymorphic DNA Technique
  • Skin / immunology
  • Skin / microbiology

Substances

  • DNA, Bacterial

Grants and funding

This research was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 063 to SEP), Consejo de Investigaciones de la Universidad Nacional de Tucumán (PIUNT 26/D 414 and 26/D 645 to SEP, and PIUNT 528), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2017-2244 to SEP, PICT 2015-2467 to CEA, and PICT2017-4324 to MENM), Ministerio de Economía y Competitividad de España (CGL2015-70070-R to JB), Fondo Nacional de Desarrollo Científico y Tecnológico (1181758 to CSA and 3180107 to AVS), Fundação de Amparo à Pesquisa do Estado de São Paulo (2016/25358-3 to LFT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.