The Influences Of Bacillus Subtilis On The Virulence Of Aeromonas Hydrophila And The Expression Of Quorum Sensing LuxS Gene

In fish farming,the bacterial disease cause high economic losses.Conventionally,the antibiotics were widely used for treating bacterial disease and preventing the disease outbreak in aquaculture industries.However,random and unscientific uses of such antibiotics create the emergence of drug-resistant,increase the tolerance of bacterial to the antibiotics,cause drug residue in fish,destroy the aquatic ecosystem.To address such issues,probiotics are introduced as a natural,eco-friendly,no drug residue substitute of antibiotics in aquaculture sectors.The properties of probiotics include improving the water quality,enhancing the immune system of the host and better growth improvement in fish.Bacillus subtilis is a kind of probiotics which is widely used in aquaculture.In our early study,we have screened out a strain of B.subtilisfrom grass carp intestinal tract which have antagonistic effect on Aeromonas hydrophila and other aquatic pathogens.The aim of this study was to explore the influence of B.subtilis on A.hydrophila.We analyzed and compared different initial inoculum concentration of B.subtilis inhibited the growth of A.hydrophila.In the co-culture,when the initial inoculum concentration of B.subtilis and A.hydrophila were 1×10~8 CFU/ml and 1×106 CFU/ml respectively,the growth of both bacteria was slowed down compared to the monocult ure.But no significant growth inhibition was observed in these two strains of bacteria.When the initial inoculum concentration of B.subtilis and A.hydrophila were 1×10~9 CFU/ml and 1×10~8 CFU/ml respectively,the growth of B.subtilis has no difference in co-culture and monoculture.The number of bacteria was significantly decreased after 4 hours of co-culture in A.hydrophila,and the growth of A.hydrophila was significantly inhibited in co-culture compared with monoculture.Therefore,we chose 1×10~9 CFU/ml B.subtilis and 1×10~8 CFU/ml A.hydrophila for subsequent experiments.The co-culture initial inoculumconcentration of B.subtilisand A.hydrophila was 1×10~9 CFU/ml and 1×10~8 CFU/ml,respectively.Thetranscription level of virulence genes of A.hydrophila and its hemolysin activityas well as its cytotoxicity wereanalyzed under A.hydrophila monoculture and co-cultured with B.subtilis.The results indicated that the transcription level of four virulence genes of A.hydrophila,including aer,ahy B,hcp,and emp,were decreased when A.hydrophila was cultured with B.subtilis.The transcription level of aer gene of A.hydrophilain co-culture was 18.01 and 3.05 fold lower than in monoculture at 6h and 8h,respectively.The transcription level of ahy B gene of A.hydrophila in co-culture was 2.07,1.43 and 3.07 fold lower compared to monoculture at 6h,8h,and 10 h,respectively.The expression level of hcp gene of A.hydrophila in co-culture was 10.99,218.48,830.28,565.48 and 481.34 fold lower compared to monoculture at 4h,6h,8h,10 h,12h,respectively.The expression level of emp gene of A.hydrophila in co-culture was 1.76 and 1.46 fold lower compared to monoculture at 6h and 8h,respectively.Furthermore,in A.hydrophila monoculture,the clear zone on blood agar plate was observed and became bigger with the increment of culture time.When A.hydrophila were co-culturedwith B.subtilis,no zone of clearance was observed,which means the extracellular productions of A.hydrophila showed attenuated hemolysin activity when A.hydrophila was cultured with B.subtilis.The cytotoxicity of A.hydrophila cultured w ith B.subtilis was monitored in CIK cells by quantifying LDH.When A.hydrophila was co-culturedwith B.subitilis,its cytotoxicity was reduced significantly compared with that of monoculture,which means the extracellular productions of A.hydrophila showed attenuated cytotoxicity when A.hydrophila was cultured with B.subtilis.Finally,the transcriptionallevel of lux S genes of B.subtilis and A.hydrophila were determined when these two species were co-cultured.RT-PCR results suggested that the transcription level of A.hydrophila was down-regulated significantly.O n the contrary,the transcription level of B.subtilis wasup-regulated significantly.These results suggested that the probiotic role of B.subtilis is related tothe inhibition of growth and virulence of A.hydrophila and quorum sensing may be involved.