Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacter...

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Bibliographic Details
Published in:Diseases of aquatic organisms 2013-03, Vol.103 (1), p.77-85
Main Authors: Bell, Sara C, Alford, Ross A, Garland, Stephen, Padilla, Gabriel, Thomas, Annette D
Format: Article
Language:English
Subjects:
Animals
Antifungal Agents - metabolism
Antifungal Agents - pharmacology
Anura
Bacteria
Bacteria - metabolism
Bacteriological Techniques
Batrachochytrium dendrobatidis
Biological Assay - methods
Cell-Free System
Chytridiomycota - drug effects
Litoria
Ranidae - microbiology
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Summary:Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.
ISSN:0177-5103
1616-1580
DOI:10.3354/dao02560