Probiotics for amphibians: advances in the selection of lactic acid bacteria for chytridiomycosis control

MARIA V. NIEDERLE; EDUARDO E. ANDRADA SUAREZ; SABRINA I. VOLENTINI; SERGIO E. PASTERIS

Roma

Conferencia; 10th Euro-Global Conference on Infectious Diseases; 2018

Statement of the Problem: Chytridiomycosis is an amphibian skin disease caused by Batrachochytrium dendrobatidis (Bd) that produces extinction around the world (1). Some amphibian-skin bacteria have been proposed as probiotic for chytridiomycosis control (2) but they excluded the lactic acid bacteria (LAB) group. Here in, we advanced in the selection of indigenous LAB from bullfrog (considered as a Bd carrier) skin to design probiotic for application during the ex situ breeding of endangered amphibians. Methodology: To determine the anti-Bd activity, co-culture assays (3) between Bd strains (CFLT 159 from Brazil; AVS4 and AVS7 from Chile) and potentially LAB isolates were performed. Isolates that previously shown exopolysaccharide (EPS) synthesis and/or autoaggregation (AA) ability (4) were evaluated for biofilm formation by using polyestyrene plates (5). Compatibility assays (4) were performed to evaluate the possibility to formulate a mixed probiotic. Findings: From 62 potentially LAB, 48 isolates had any anti-Bd activity. The 16s RNA sequence analysis allowed obtaining 97-99% of identity that matches with Enterococcus and Lactobacillus. Thus, Enterococcus sp. 90, 564, 747, 762; Lactobacillus sp. 10, 529, and Enterococcus gallinarum CRL 1826 (previously characterized) inhibited all the Bd strains. Three LAB isolates exhibited low biofilm formation, while E. gallinarum showed moderate production. This ability was not always associated with AA or EPS synthesis. The compatibility assays indicated that the LAB isolates could be included in mixed probiotic with the exception of Enterococcus sp. 742 that was inhibited by E. gallinarum. Conclusion & Significance: E. gallinarum CRL 1826 resulted the best strain for a probiotic since it has many beneficial properties: anti-Bd activity, AA, EPS synthesis, biofilm formation, medium hydrophobicity and GRAS properties according to in vitro and in vivo tests (3-7). However, Enterococcus sp. 747 would maximize some probiotic properties of the CRL strain; therefore, a mixed probiotic can be proposed.References1. Rosa GM, Sabino-Pinto J, Laurentino TG, Martel A, Pasmans F, Rebelo R, Griffiths RA, Stöhr AC, Marschang RE, Price SJ, Garner TW, Bosch J (2017) Impact of asynchronous emergence of two lethal pathogens on amphibian assemblages. Scientific Reports 7: 43260.2. Woodhams DC, Bell SC, Caprioli RM, Chaurand P, Lam BA, Reinert LK, Stalder U, Vazquez VM, Schliep K, Hertz A, Rollins-Smith LA (2016) Life history linked to immune investment in developing amphibians. Conservation Physiology 4(1): cow025.3. Niederle MV, Ale CE, Acevedo MA, Nader-Macías ME, Toledo LF, Pasteris SE (2016) Lactic acid bacteria as potentially control agents of Batrachochytrium dendrobatidis. International Simposium on Lactic Acid Bacteria (SIBAL). Tucumán. Argentina.4. Montel Mendoza GM, Pasteris SE, Ale CE, Otero MC, Bühler MI, Nader-Macías ME (2012) Cultivable microbiota of Lithobates catesbeianus and advances in the selection of lactic acid bacteria as biological control agents in raniculture. Research in Veterinary Science 93(3):1160-1167.5. Leccese Terraf MC, Juárez Tomás MS, Nader-Macías ME, Silva C (2012) Screening of biofilm formation by beneficial vaginal lactobacilli and influence of culture media components. Journal of Applied Microbiology 113(6):1517-1529.6. Montel Mendoza G (2014). Doctoral Thesis. Estudios fisiológicos, funcionales y tecnológicos de bacterias lácticas en ranario. Bases para el diseño de productos probióticos para la prevención de infecciones. Universidad Nacional de Tucumán. Argentina.7. Pasteris SE, Montel Mendoza G, Llano RJ, Pucci Alcaide F, Nader-Macías MEF (2017) Preliminary assessment of in vivo safety of potentially probiotic lactic acid bacteria for American bullfrog culture. Aquaculture Research 48(5): 2157-2172.