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

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.

Functional probiotics of lactic acid bacteria from Hu sheep milk

“…The hydrophobicity of the bacterial surface was determined by the Microbial Adhesion to Hydrocarbon (MATH) assay [54]. Bacterial hydrophobicity was measured through using different organic solvents such as n-dodecane (Aladdin Co., Ltd. Shanghai, China), xylene (Sinopharm Chemical Reagent Co., Ltd. Shanghai, China), and chloroform (Sinopharm Chemical Reagent Co., Ltd. Shanghai, China).…”

“…A t stands for the value of OD 600 after mixing. The degree of bacterial hydrophobicity was classified as low (0-29%), medium (30-59%) or high (60-100%) [54].…”

Skin microbiome correlates with bioclimate and Batrachochytrium dendrobatidis infection intensity in Brazil’s Atlantic Forest treefrogs

“…Skin microbiomes constitute a critical component of the amphibian immune system 30 , 68 because certain bacterial taxa are capable of producing anti-fungal metabolites that can suppress growth of Bd 33 , 69 – 71 and, more generally, some bacterial taxa compete with each other, which limits the ability of multiple pathogens to infect 20 . Consequently, susceptibility to Bd might differ in amphibians across the Atlantic Forest if the skin bacterial microbiome is shaped differently due to heterogenous abiotic factors (reviews in 2 , 20 , 72 – 76 ).…”

Composição da comunidade bacteriana cutânea em anfíbios na região Oeste do Pará, Amazônia, Brasil

“…de anfíbios, afirmaram que estas bactérias são benéficas e possuem propriedades para serem utilizadas pela indústria como probiótico. Dessa forma, é possível pressupor que este gênero faz parte da microbiota natural dos anfíbios e que pode agir como agente benéfico para estes animais, além das aplicações biotecnológicas (NIEDERLE et al, 2019).…”