Computational protocol: Interacting Symbionts and Immunity in the Amphibian Skin Mucosome Predict Disease Risk and Probiotic Effectiveness

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Protocol publication

[…] To determine the effects of competitive interactions and temperature on probiotic potential, 11 common host-associated isolates were chosen. These included two isolates of Serratia plymuthica and one isolate of Janthinobacterium lividum from egg clutches of midwife toads, three isolates of Flavobacterium johnsoniae and five species of Pseudomonas isolated from the skin of adults. Based on 16S rRNA gene sequences, all 11 isolates were considered unique operational taxonomic units (OTUs) at 99%, but clustered into 7 OTUs at 97% similarity as determined by the UCLUST algorithm in QIIME. The 16S rRNA gene sequences of all isolates were deposited in the European Nucleotide Archive (Table S1 in ).In one set of experiments, bacterial isolates were freshly grown at 18°C on RIIA agar media supplemented with 1% tryptone then transferred to experimental conditions. Bacteria and Bd (Swiss isolate TG 739) readily grew on the same media. Plate experiments were performed in duplicate. Both isolates of Serratia plymuthica were grown separately at 18 and 25°C, or at 18°C on media inoculated with Bd and allowed to dry before streaking the bacteria. Two isolates of F. johnsoniae were grown separately or combined on media inoculated with Bd, and grown at 18°C. When combined, each isolate was streaked across the entire plate. Three Pseudomonas isolates were grown either separately, combined, or combined on media inoculated with Bd, and grown at 18°C. Control plates of sterile media or Bd-only were also tested. All plates were incubated for 3 days, and then rinsed with 2 ml sterile Mili-Q water. Rinse water was then filtered through a 0.22 µm syringe filter.Bacteria were also grown in liquid RIIA media for 4 d at 14, 19, and 22°C, and metabolites filtered as above. Metabolites from liquid cultures were added to Bd zoospores (Global panzootic lineage VMV 813 from a bullfrog, Lithobates catesbeianus tadpole) to test for inhibitory effects on pathogen growth. To determine the effect of bacterial filtrate on Bd growth, Bd zoospores were harvested in 1% tryptone and counted under a hemocytometer. Wells of a 96-well plate were inoculated with 50 µl zoospores at 8×106 zoospores per ml. Then, 50 µl of filtrate (or filtrate diluted 1∶10) from each of the experimental or control plates, or liquid cultures, was added to the wells in replicates of four. In addition, 6 positive control wells contained Bd and 50 µl sterile water or RIIA media, and 6 negative control wells contained heat-killed Bd and 50 µl sterile water or RIIA media. The change in optical density measured at 490 nm absorbance over 7 days growth at 19°C was recorded using a Victor3 multilabel plate reader (PerkinElmer). Standard statistical testing was carried out in IBM SPSS Statistics 22. Significant Bd growth inhibition (or enhancement) caused by bacterial filtrate was determined by t-test, and a repeatable result (Table S2 in ). Percent inhibition depended on filtrate dose (see ) and was not considered comparable among bacterial isolates. […]

Pipeline specifications

Software tools UCLUST, QIIME, SPSS
Databases ENA
Application Miscellaneous
Organisms Homo sapiens, Fungi, Batrachochytrium dendrobatidis
Diseases Infection, Zoonoses