Detection Of The Exotoxin Of Vibrio Mimicus And Study On The Bacteriostasis, Antitoxin Activities Of Chinese Herbal Medicine

Vibrio mimicus is a common pathogenic vibrio,it not only causes serious ascitic fluid disease of fish and Eriocheir sinensis,but also leads to human diarrhea and food poisoning.The primary means to prevent and treat the disease is to use antibiotics currently,bacterial resistance and drug residues leading to the security problems of aquatic products are highlighted with the repeated use of antibiotics.In this study V. mimicus isolated in Anhui is taken as the target bacterium.In order to select the best Chinese herbal compound,provid materials to the following clinical trial research and lay the foundation for exploiting herbal preparations for the treatment of ascitic fluid disease,the bacteriostasis and antitoxin activities of Chinese herbal compound were studied systematically on the basis of revealing drug resistance of four strains and their exotoxic pathogenicity.Firstly,exotoxic gene(vmhA,hlyA,ctxA,zot,ace)from V.mimicus isolated in Anhui and exotoxic pathogenicity were detected.Primers were designed based on the sequence of V.mimicus or V.cholera exotoxic gene,exotoxic gene were amplified by PCR from bacterial genomic DNA and constructed recombinant plasmid and then sequenced and analyzed.The results showed that four isolated strains all carded exotoxic gene,but virulence genotypes were different in the different isolated strains. Virulence genotypes of HX4 strain,04-13 strain,04-14 strain and 04-5 strain were vmhA~+hlyA~-ctxA~-zof~-ace~-,vmhA~-hlyA~+ctxA~-zot~-ace~-,vmhA~-hlyA~+ctxA~-zot~+ace~- and vmhA~-hlyA~-ctxA~-zot~+ace~- respectively.The vmhA fragment from HX4 strain was 1 391 bp in length and the nucleotide homology of vmhA was up to 97.3%with the corresponding nucleotide of only foreign V.mimicus referent strain VMU68271.The hlyA fragment from 04-13 strain and 04-14 strain were all 934 bp in length and the nucleotide homology were 92.4%～99.3%between referent strains isolated from home and abroad.On the basis of exotoxin extraction by protein separate technology,the hemolytic activity of exotoxin was detected by blood agar diffusion method and microdilution method,exotoxic pathogenicity was detected by animal experiments.The results showed the exotoxin of four strains V.mimicus were all had strong enterotoixicity and pathogenicity to mice and grass carp.HX4,04-13 and 04-14 strains all had hemolytic activities except 04-5 strain,their hemolytic titre were between 1:64 and 1:256.The results suggested that exotoxin played an important role in the pathogenic process. Secondly,the drug resistances of V.mimicus isolated in Anhui to antibiotics were detected.In this study the drug resistances of four strains to nineteen kinds of antibiotics were detected by K-B disc agar diffusion method.The results showed that they had different levels of resistance toβ-lactams,aminoglycosides,tetracyclines, macrolides and quinolone antibiotics,they had multiple drug resistances.Finally,the effect of Chinese herbal medicine on bacteriostasis,antitoxin activities and intestinal microflora and advantageous flora of grass carp were detected systematically.Bacteriostasis activities of 27 kinds of single-flavor Chinese herb were assayed by Plat stiletto method in vitro.The intermediary sense and high sense Chinese herbal medicines were selected and their minimal inhibitory concentration(MIC)were detected by improved microdilution method.The results showed that Rhizoma coptidis,Rheum officinale,Flos lonicerae and Cortex fraxini achieved high sensitivity and their MIC were 1.96～62.72 g/L,followed by Spica prunellae,Euphorbia humifusa,Cortex Moutan,Fructus forsythiae and Cortex phallodendri,they achieved intermediary sensitivity and their MIC were 31.36～250 g/L.Chinese herbal medicine in above as main medicine were combined to five compounds with Poria cocos,Semen plantaginis as adjunctive medicine,Radix astragali,Glycyrrhiza uralensis as guide medicine and their bacteriostasis activities in vitro and indirect antagonistic effect to exotoxin were detected.The results showed that compound 5 had the best bacteriostasis activities in vitro and their MIC were all 1.96 g/L to four strains V.mimicus;followed by compound 1 and 3,their MIC were from 1.96 to 7.84 g/L;compounded 2 and 4 had the lower bacteriostasis activities,MIC were 31.36～125 g/L.Compound 5 had the strongest indirect antagonistic effect to exotoxin, followed by compound 2,3 and 4,compound 1 had no antagonistic exotoxin activities. It determined that compound 5 had the best compatibility effect with a better strong bacteriostasis activities and indirect antitoxin secretion role in vitro by integrated consideration.Antibacterial activities of compound 5 in vivo was detected by artificial infection experiment and the effect on intestinal microflora and advantageous flora of grass carp were measured by bacteriological methods.The results showed that compound 5 could protect experimental grass carp from V.mimicus infecting through wound and reduce intestinal microflora and inhibit growth of intestinal conditional pathogenic Aeromonas and Acinetobacter.Above all,this study successfully screened out Chinese herbal compound with bacteriostasis activities in vitro,antibacterial activities in vivo,indirect antagonistic effect to exotoxin and immunoregulating activities on the basis of revealing drug resistance of V.mimicus and pathogenicity of exotoxin.The study results can provide materials to the following clinical trial research,also lay the foundation for exploiting herbal preparations for the treatment of ascitic fluid disease.