Ethanolic extract of red seaweed Gracilaria fisheri and furanone eradicate Vibrio harveyi and Vibrio parahaemolyticus biofilms and ameliorate the bacterial infection in shrimp.

Bacteria respond to host immunity for their proliferation and survival by cell-cell communications such as biofilm formation, bioluminescence, and secreting virulence factors. In the biofilm form, bacteria are more resistant to various antimicrobial treatments and withstand the host's immune system. The approaches of deciphering biofilm formation for treating bacterial infections are therefore highly desirable. Recently, we have reported that the ethanolic extract of the red seaweed Gracilaria fisheri (G. fisheri) enhanced immune activities and inhibited growth of the luminescent bacteria Vibrio harveyi in shrimp. We undertook the present research study in order to evaluate and compare the effectiveness of the ethanolic extract from G. fisheri and furanone, a known biofilm inhibitor, in inhibiting the formation of clinically important Vibrio biofilms. The results showed that sub-lethal concentrations of both the ethanolic extracts (5, 10 and 100 µg ml-1) and furanone (5 µM) inhibited biofilm formation by V. harveyi and Vibrio parahaemolyticus and also light production (luminescence) in V. harveyi. It is known that V. harveyi mediated light production via autoinducer AI-2 pathway, we further determined whether the inhibitory effect of the extract was involved the AI-2 signaling. The bioluminescence assay was conducted in an AI-2 deletion mutant V. harveyi. Supplementation of the AI-2 containing media with the extract or furanone impaired the light production in the mutant V. harveyi suggesting that the extract interfered AI-2 mediated light production similar to furanone. In vivo challenge study showed that the low concentrations (Sub MICs) of the ethanolic extract and furanone decreased bacterial adhesion and colonization in the surfaces of stomach lumen, down-regulated expression of a virulence factor, and protected shrimp against mortality from V. harveyi and V. parahaemolyticus infection. In conclusion, the present results suggest a potential application of the low concentrations of the ethanolic extract of G. fisheri as an efficient approach for treating biofilm-associated Vibrio diseases in aquacultures.

Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection.

Many bacteria, including Vibrio pathogens of shrimp, need to colonize and/or form biofilms in hosts or the environment to cause disease. Thus, one possible control strategy for shrimp Vibriosis is biofilm inhibition. With this objective, an extract from the Japanese fermented soybean product, Natto was tested with the luminescent shrimp pathogen Vibrio harveyi (VH) for its ability to inhibit or degrade biofilm and to interfere with cell growth in broth. Natto is a traditional fermentation product of Bacillus subtilis var Natto (BSN1). Using 96 well microtiter plates coated with 0.4% chitosan, we found that biofilm formation by VH was inhibited, while growth in parallel broth cultures was not. When an extract from Natto prepared using BSN1 was mixed with feed for the whiteleg shrimp Penaeus vannamei before immersion challenge with V. harveyi at 106 cfu/ml, survival was significantly higher (p≤0.05) than for control shrimp given feed without these additives. Further work done to test whether d-amino acids were involved in biofilm formation as previously reported for B. subtilis, Staphylococus aureus and Pseudomonas aeruginosa gave negative results. In conclusion, we discovered that Natto extract can inhibit Vibrio biofilm formation and that it or BSN1 alone added to shrimp feed can significantly reduce shrimp mortality in immersion challenges with pathogenic VH. This shows some promise for possible application against Vibriosis in shrimp since Natto is generally regarded as safe (GRAS) for human consumption.