Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay
Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay
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DOI：
10.3354/dao02560
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发表时间：
2013-03-13
影响因子：
1.4
通讯作者：
Thomas, Annette D.
中科院分区：
文献类型：
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作者：
Bell, Sara C.;Alford, Ross A.;Thomas, Annette D.
青蛙皮肤上存在的某些细菌可以预防病原真菌 Batrachochytrium dendrobatidis (Bd) 的感染，从而赋予其抗病能力。先前的研究使用基于琼脂的体外激发试验来筛选细菌的 Bd 抑制活性，并确定细菌补充试验的候选者。然而，基于琼脂的测定可能难以建立和可靠地复制。为了克服这些困难，我们开发了一种半定量分光光度挑战测定技术。从过滤的细菌培养物中制备无细胞上清液，并将其添加到 96 孔板的重复孔中，其中含有悬浮在胰蛋白胨-明胶水解产物-乳糖 (TGhL) 肉汤培养基中的 Bd 游动孢子。然后每天在分光光度计上读取板的读数，直到阳性对照达到最大生长以确定 Bd 的生长曲线。我们通过筛选澳大利亚绿眼树蛙 Litoria serrata 的皮肤细菌来测试该技术。在测试的细菌中，31% 表现出一定程度的 Bd 抑制作用，而一些细菌可能促进 Bd 生长，这是一种以前未知的效果。我们的无细胞上清挑战测定技术是一种有效的体外方法，用于筛选细菌分离株是否具有强 Bd 抑制活性。它有助于扩大生物强化研究领域，可以在减轻壶菌病对世界各地两栖动物的影响方面发挥重要作用。
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.