Modification of an agar well diffusion technique to isolate yeasts that inhibit Vibrio parahaemolyticus , the causative agent of acute hepatopancreatic necrosis disease

Abstract

Vibrio parahaemolyticus is the causative agent of acute hepatopancreatic necrosis disease. In this study, we isolated yeasts possessing inhibitory activity against V. parahaemolyticus. In general, the generation time of yeasts is longer than that of bacteria. Therefore, the techniques used to evaluate yeasts with activity against bacteria using the same incubation time may not be appropriate. In this work, an agar well diffusion technique was modified to resolve this problem. Various selected yeast colonies were selected and each colony was introduced into an agar plug and incubated for 48 hr at room temperature. V. parahaemolyticus was spread onto Mueller Hinton agar plates, after agar plugs were removed to form wells and replaced with agar plugs seeded with yeast. After incubating for 24 hr at room temperature, the diameter of the inhibition zone was recorded. A total of 22 yeast isolates exhibited inhibitory activity towards V. parahaemolyticus. Thirteen isolates that possessed high inhibitory activity were identified by PCR and confirmed by sequencing, with four genera and 11 species of yeast identified. In a Galleria mellonella larvae model, a selected yeast strain reduced the larval mortality rate after being challenged with V. parahaemolyticus, indicating the usefulness of this yeast in shrimp aquaculture.