Antimicrobial Activity And Mechanisms Of Essential Oils Against Pathogenic Bacteria Isolated From Farmed Turbot Scophthalmus Maxius

Turbot,Scophthalmus maximus(L.),is a traditionally marine-farmed fish species,which was introduced into Chinain 1992.Following the breakthrough in artificialbreeding,the culture of turbot on a commercialbasis spread rapidly,and is becoming one of themost economically important species in coastal areas of northern China.However,intensive turbot farming in China has resulted in growing problems of bacterial diseases.Currently,the pathogenic bacteria are mainly controlled by use of antibiotics,which bring bacterial resistance,aquatic environmental pollution and drug residues problems.Therefore,it is highly necessary to develop an alternative antimicrobial agents to control the bacterial infections in farmed turbot.Essential oils are volatile and aroma compounds and are formed by aromatic plants as secondary metabolites.Essential oils possess proven antimicrobial activities,and have received attention as novel types of antimicrobial agents to treat and control diseases in aquaculture instead of commercial antibiotics.The main contents of this study are summerized as follows:(1)Isolation and identification of the pathogenic bacteria isolated from the farmed turbot and investigation of their antimicrobial susceptibility.Thirty-three strains of bacteria were isolated from the turbots suffering from swollen abdomen,reddish dody syndrome,exophthalmia disease and red mouth symptom in Dalian,and identified by morphological characteristics,physiological and biochemical characteristics and the sequence similarity analysis of the 16 S rDNA genes.The results showed that a total of 33 strains of bacteria were identified as 29 strains of Edwardsiella tarda,one strain ofVibrio scophthalmi and three strains of Citrobacter freundii.Antimicrobial susceptibility tests of isolates were detected by the routine method of K-B using 30 antimicrobial agents,and the isolated strains showed muti-drug resistance and different antimicrobial resistance pattern.(2)To evaluate the antibacterial activity of essential oils against pathogenic bacteria isolated from farmed turbot Scophthalmus maxius in vitro.The paper disc diffusion method and microbroth dilution technique were employed to determine the antimicrobial activity of 16 essential oils.Cell membrane disruption induced by Litsea cubeba oil was examined by transmission electron microscopy(TEM),and by measuring the release of intracellular lactate dehydrogenase(LDH)and 260 nm absorbing material.The 14 essential oils showed different antimicrobial activities against V.scophthalmi HZ-C1,and cinnamaldehyde,thymol and carvacrol had the highest activity with a minimum inhibitory concentration(MIC)of ≤1 μL/mL,followed by citral,lemongrass and Litsea cubeba oil with a MIC of 0.25-4 μL/mL.Litsea cubeba oil can disrupt the plasma membrane of V.scophthalmi HZ-C1 and cause the leakage of protease and nucleic acid.(3)In vivo,the antimicrobial activity of Litsea cubeba oil in vivo were investigated using the turbot model of E.tarda(1×10~7CFU/fish),V.scophthalmi(2×10~7CFU/fish)and C.freundii(1×10~7CFU/fish)by intraperitoneal injection.After immersing in a bath containing 200 μL/L Litsea cubeba oil,the mortality of turbots infected by E.tarda,V.scophthalmiand C.freundiiwas reduced from 60%,50% and 40% to 20%,0% and 20% respectively.Additionally,the antimicrobial activity of Litsea cubeba oil in vivo werealso investigated using theZebrafish Model.After treatment by 200 μL/L Litsea cubeba oil,the mortality of Zebrafish infected by E.tarda andC.freundiiwas reduced from 60%,90% to 10% and 20% respectively.In conclusion,this study demonstrated that E.tarda,V.scophthalmi and C.freundiiwerethe major pathogens infecting reared turbot in Dalian,and all the isolated strains showed multi-drug resistance.Additionally,cinnamaldehyde,thymol,carvacrol and essential oils rich in citral show high antibacterial activities against the isolated pathogenic bacteria from farmed turbot,and thus have the potential to replace the use of antibiotics for treating bacterial disease.This study provide an etiological evidence for control of bacterial disease in cultured turbot in Dalian and offer a new strategy to effectively reduce the antibiotic use in turbot aquacuture.