Vibrio biofilm inhibitors screened from marine fungi protect shrimp against acute hepatopancreatic necrosis disease (AHPND)

Abstract

Marine fungi are known to produce many secondary metabolites with diverse biological activities, including antibacterial activity. We screened cell-free culture broths of 39 different isolates of obligate marine fungi for inhibitors of growth and biofilm formation with 7 isolates of Vibrio bacteria that originated from shrimp ponds affected by luminesent shrimp disease or acute hepatopancreatic necrosis disease (AHPND). Using microtiter-plate growth and biofilm inhibition assays, inhibitory effects for each fungal isolate varied depending on the Vibrio isolate. Overall, broth from 11 out of 39 isolates of the fungi examined inhibited biofilm formation by 3HP plus 4 to 6 of the other Vibrio isolates tested. Overall, for most fungal isolates, Vibrio biofilm inhibition activity was greater than growth inhibition activity. Based mainly on high inhibition activity against 3HP biofilm formation, but also on inhibition of at least 4 out of 6 of the other Vibrio isolates tested, culture broths of 4 fungal isolates (AK112, MCR1, MCR55 and MCR113) were studied further as possible feed additives to protect post larvae (PL) of Penaeus vannamei against AHPND. Specifically, the cell-free, broth-supplemented feed was administered for 7 days before the PL were challenged with 3HP by immersion at 106 CFU/ml followed by further use of the same supplemented feed for another week during which shrimp health and mortality were monitored. Survival in the unchallenged, negative-control group fed supplemented feed was approximately 88% while survival in the challenged shrimp group receiving feed supplemented with cell-free broth from MCR55 was (70%) and not significantly different from that in the negative control group (p > .05). By contrast, survival of the challenged, positive control groups fed with either un-supplemented feed or feed supplemented with culture medium was ≤18%. The fungal isolates had been identified by published methods based on morphology, and 36 out of 39 were confidently identified to at least genus level. Isolate MCR55 that protected shrimp against AHPND was identified as Oceanitis cincinnatula from the Order Microascales, Class Sordariomycetes. The results obtained indicated that the culture broth of O. cincinnatula contained a substance(s) that did not inhibit the growth of 3HP but could potentially be used as shrimp feed supplement to protect shrimp against AHPND, possibly by inhibiting biofilm formation in the shrimp stomach.