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. KEY WORDS: Chytridiomycosis · Challenge assay · Bacterial metabolite · Amphibian disease · BioaugmentationResale or republication not permitted without written consent of the publisher

Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability

“…At approximately 3 days, most bacterial cultures reached the later phases of growth, when inhibitory metabolites are produced (95). This is a commonly accepted practice and is currently the best method for testing a large number of bacterial isolates (35,60,93,94).…”

“…dendrobatidis challenge assays with each bacterial isolate (n ϭ 341) and B. dendrobatidis isolate JEL 404 (Maine, USA) using a modified version of a protocol by Bell et al (93). To begin, we grew cryopreserved bacteria on 1% tryptone plates for 3 days, passaged the bacteria in 3 ml of 1% tryptone broth, and grew the bacteria on a shaker at 100 rpm for an additional 3 days.…”

“…We grew these cultures on a shaker at 100 rpm for 3 days. To obtain microbially produced metabolites, termed cell-free supernatant (CFS [93]), we centrifuged the bacterial isolate/B. dendrobatidis cocultures, B. dendrobatidis monocultures, and negative controls at 10,000 rpm for 5 min.…”

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

“…We tested each isolate's ability to inhibit Bd with a spectrophotometric assay (similar to Bell et al [27]). We identified each isolate taxonomically by sequencing the 16S rRNA gene [24] and analysing the sequence with the Ribosomal Database Project's Sequence Match tool (http://rdp.…”

Amphibian Symbiotic Bacteria Do Not Show a Universal Ability To Inhibit Growth of the Global Panzootic Lineage of Batrachochytrium dendrobatidis

“…The inclusion of the size of the bacterial streak in this data conversion step ensured that bacterial density was controlled for in the analyses. An alternative method of quantifying B. dendrobatidis inhibition using 96-well plates may be more accurate and quantifiable than plate challenges, but it does not allow consideration of the direct competition (e.g., for space and resources) that may occur between B. dendrobatidis and bacteria and that may also occur on the skin of amphibians (22).…”