Studies On The Mechanism Of Inhibition Of Vibrio Parahaemolyticus By Combination Of Herbal Extracts And Antibiotics

Vibrio parahaemolyticus is a gram-negative halophilic pathogen that is often found in sea water,fish,shrimp,shellfish and other marine products near the coast.V.parahaemolyticus can lead to Early Mortality Syndrome（EMS）of penaeus and cause great losses to aquaculture in Asia and China.In recent years,the abuse of antibiotics in aquaculture resulted in the emergence of drug-resistant strains of V.parahaemolyticus and the failure of many conventional antibiotics,which significantly endangered the edible safety of aquatic products and the health of the people.In this paper,the synergistic antibacterial effects and the mechanisms of various herbal extracts and aquatic antibiotics against V.parahaemolyticus were studied.The result was applied in the culture of Exopalaemon carinicauda and the prevention and cure mechanisms of V.parahaemolyticus disease were explored.This paper opens up a new idea for the prevention and cure of aquaculture vibriosis.The main research results are as following:1.In vitro antibacterial effects of herbal extracts and antibiotics on V.parahaemolyticusFour groups of herbal extracts and antibiotics having synergistic antibacterial effects on V.parahaemolyticus were screened out.The optimal ratio were 1/8 MIC_sealwort:1/4 MIC oxytetracycline、1/4 MICpepper:1/4 MIC oxytetracycline、1/4 MIChawthorn:1/4 MIC oxytetracycline、1/4MIC_licorice:1/8 MIC _{oxytetracycline}.By exploring the antibacterial mechanisms,it was found that pepper,sealwort and hawthorn could improve the membrane permeability of V.parahaemolyticus,while Sealwort,hawthorn and Glycyrrhiza uralensis could increase cell wall permeability,respectively.We compared the effects of oxytetracycline,pepper and sealwort on the expression of efflux pump related genes in V.parahaemolyticus.The results showed that the expression of vp1998（tolC）and vp1999 were up-regulated for oxytetracycline,sealwort and pepper,and increased by 151.91 times and 94.84 times respectively especilally for oxytetracycline.In addition,the expression of RND family transporter,outer membrane protein and ion pump gene were all up-regulated for pepper as well as oxytetracycline,except vpa0096,vpa1186 and vp1178.For sealwort,21 genes were down regulated except for vp1998 and vp1999.The synergistic antibacterial effects of oxytetracycline,sealwort,pepper,hawthorn and licorice disappeared after adding multidrug resistant pump inhibitor CCCP.2.Knockout of efflux pump related genes of V.parahaemolyticus and its correlation with combined bacteriostasisVp:△tolC,Vp:△1993 and Vp:△1999 were successfully constructed to explore the synergistic mechanism of ABC efflux pump in oxytetracycline and herbal extracts.By comparing the growth curve,acid tolerance,bile tolerance and drug resistance of mutant and wild strain（WT）,the results showed that the growth curve and acid tolerance ability of V.parahaemolyticus were not related to tolC,vp1993 and vp1999.The bile tolerance of Vp:△tol C was significantly decreased compared with WT.The expression of tolC and vp1999 genes could improve the membrane permeability of V.parahaemolyticus.The sensitivity to erythromycin of the Vp:△tol C was significantly higher than the WT.The combined antibacterial effects of oxytetracycline and sealwort on WT and mutant strains were compared,and no significant difference was found.3.Prevention and control of vibrio disease of Exopalaemon carinicauda by combination of herbal extracts and antibiotics（1）The culture conditions of Exopalaemon carinicauda were as follows:25℃,salinity 20 degrees,pH 8.0 and culture density 30 tails/basin.The concentration of immersion infection of V.parahaemolyticus was 1.0×10⁸ CFU/m L.（2）The combination of oxytetracycline and hawthorn could improve the control ability of oxytetracycline on vibriosis of Exopalaemon carinicauda.The activities of pepsin,cellulase,immune enzyme and lysozyme increased in combination group（oxytetracycline and Hawthorn）,and the activities of pepsin,AKP and lysozyme were significantly higher than those of oxytetracycline group（p<0.05）.The expression of Toll receptor gene in the combined group was significantly up-regulated than the oxytetracycline group（p<0.05）.