title: Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.
abstract: 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.
authors.affiliation: School of Marine and Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia. sara.bell@my.jcu.edu.au
authors.forename: Sara C
authors.initials: SC
authors.lastname: Bell
authors.forename: Ross A
authors.initials: RA
authors.lastname: Alford
authors.forename: Stephen
authors.initials: S
authors.lastname: Garland
authors.forename: Gabriel
authors.initials: G
authors.lastname: Padilla
authors.forename: Annette D
authors.initials: AD
authors.lastname: Thomas
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology*; D001419:Bacteria / Q000378:metabolism*; D001431:Bacteriological Techniques; D001681:Biological Assay / Q000379:methods; D002474:Cell-Free System; D008411:Chytridiomycota / Q000187:drug effects*; D011898:Ranidae / Q000382:microbiology*
chemical_list: D000935:Antifungal Agents