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

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.

Genomic epidemiology of the emerging pathogen Batrachochytrium dendrobatidis from native and invasive amphibian species in Chile.

Emerging fungal diseases represent a threat to food security, animal and human health worldwide. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been associated with catastrophic and well-documented amphibian population declines and extinctions. For the first time, Bd was cultured from native and non-native wild amphibians in Chile. Phylogenomic analyses revealed that Chilean isolates AVS2, AVS4 and AVS7 group within the global panzootic lineage of Bd (BdGPL) in a single highly supported clade that includes a genotype previously isolated from the United Kingdom. Our results extend the known distribution of BdGPL in South America and suggest a single and relatively recent introduction of BdGPL into the country, providing additional support to the role of anthropogenic activity in the global spread of this panzootic lineage.

Non-invasive sampling methods for the detection of Batrachochytrium dendrobatidis in archived amphibians.

Chytridiomycosis, an emerging infectious disease of amphibians caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is associated with amphibian population declines worldwide. Investigation of the origin and spread of the pathogen requires examination of archived museum specimens of amphibians. Examination for Bd infection is usually done using histological techniques, which are often too destructive for valuable museum material. Three alternative methods for Bd detection (skin swabbing, brushing and scraping) were evaluated for ability to yield Bd DNA and destructiveness to specimens. Archived amphibians known to be Bd positive and which had been preserved in either formalin or ethanol for many years were used. Samples were analysed using a Bd-specific quantitative real-time Taqman PCR (qPCR) assay. There was no difference in the ability of each of the techniques to detect Bd infection, with the pathogen being detected in 75 to 81% of the 16 ethanol-fixed frogs examined. Visible evidence of sampling was left by scraping, but not by swabbing or brushing. The brush-qPCR technique detected higher counts of genomic equivalents than the other 2 sampling methods, although differences were not statistically significant. The qPCR assay did not detect Bd from any of the 6 formalin-fixed frogs examined, regardless of the sampling method. Nondestructive sampling techniques enable qPCR analysis of ethanol-preserved museum specimens for Bd. Recently, the incorporation of DNA cleanup steps allowed the detection of Bd in destructively sampled tissues from formalin preserved specimens. Further studies using nondestructive sampling incorporating DNA cleanup steps for the detection of Bd in formalin preserved specimens are warranted.