Quorum quenching Bacillus spp.: an alternative biocontrol agent for Vibrio harveyi infection in aquaculture.

Quorum sensing (QS) is a type of cell to cell communication in bacteria that can also regulate the virulence potential in pathogenic strains. Hence, QS disruption, i.e. the quorum quenching (QQ) mechanism, is presently being explored as a novel bio-control strategy to counter bacterial infections. In the present study, we characterized the QQ ability of Bacillus spp. strains to reduce the expression of some virulence factors of a shrimp pathogen, Vibrio harveyi. We screened a total of 118 spore-forming bacterial isolates from aquaculture ponds and mangrove soil for their ability to degrade the synthetic N-acyl-homoserine lactones (AHLs) C4-HSL, C6-HSL, C8-HSL, and C10-HSL. We then selected the top 17 isolates with high AHL-degradation ability for further study. Among them, B. subtilis MFB10, B. lentus MFB2, and B. firmus MFB7 had the highest ability for degradation. These 3 isolates suppressed the expression of virulence genes encoding protease, lipase, phospholipase, caseinase, chitinase, and gelatinase, and potentially inhibited the biofilm formation of V. harveyi MFB32. The reduction in expression of virulence genes like those coding for metalloprotease, serine protease, and haemolysin were confirmed by real-time PCR analysis. Moreover, in an in vivo challenge experiment, these Bacillus spp. protected Penaeus monodon post-larvae against V. harveyi MFB3 infection. Our results demonstrate the potential application of AHL-degrading Bacillus spp. as an alternative to antibiotics in shrimp hatcheries to control luminescent vibriosis. This novel bio-therapeutic method is a promising approach towards disease control in shrimp aquaculture.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon.

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

Strain variability and pathogenic potential of tdhcarrying Vibrio parahaemolyticus isolated from diseased Penaeus monodon.

A disease outbreak in 42-d-old black tiger shrimp Penaeus monodon juveniles from a commercial aquaculture farm in Kerala, India, was investigated. The cause of the disease outbreak was confirmed as Vibrio parahaemolyticus by biochemical tests, PCR targeting the toxR gene and pathogenicity testing of the isolates. All of the isolates tested negative by PCR specific for V. parahaemolyticus associated with acute hepatopancreatic necrosis disease (AHPND), implicating vibriosis unrelated to AHPND as the cause of mortality. Among the 19 isolates obtained, 2 possessed the tdh gene (coding for thermo-stable hemolysin), whereas none of the isolates possessed trh. The LD50 value of 8 isolates of V. parahaemolyticus from diseased and apparently healthy shrimp ranged from 2.7 × 104 to 4.9 × 105 CFU ml-1 by immersion challenge of P. monodon postlarvae. BOX-PCR and dendrogram analysis of the bacterial isolates revealed that the isolates from moribund and apparently healthy shrimp formed separate clusters, indicating that these isolates originate from separate clones. The isolates from moribund shrimp including tdh-positive V. parahaemolyticus clustered together. The present study represents the first report of tdh-positive V. parahaemolyticus causing disease in a shrimp farm.