| Aeromonas hydrophila is of worldwide concern and posting serious hazard on aquaculture and human health.To date,A.hydrophila is mainly controlled by antibiotics.In China,more than 5,000 tons of tetracycline antibioticsare used in aquacultural industries annually,including oxytetracycline(OXY),tetracyclines and aureomycin.OXY,as a high efficient,low toxic and rapidly metabolized,is directly added to aquatic feeds as an aqua drugs.The abuse of OXY resulted in large amount of oxytetracycline tolerant strains,which significantly reduces the efficiency of the antibiotic.Due to the high cost of new drug developments,increase the efficiency of the available drugs become more and more important.Previous researches on A.hydrophila generally focused on isolation of genes related to tetracycline-antibiotics resistance and developments of new vaccines.However,studies on the relationship between stress and acquired tolerances,and the relationship between virulence factors and drugs resistance are very limited.This study investigated the stress and acquired OXY resistances of A.hydrophila.A OXY resistant strain(OXY-R)was induced from A.hydrophila(ATCC 7966;referred as Ah in this thesis)by low dose OXY.Whole genome resequencing and mass spectrum analyses of Ah and OXY-R strains were performed which unravel the genetic background of OXY-Resistance.In addition,the author also investigated the OXY-induced holoproteins and membrane proteins differentiate expressions of Ah under different OXY concentration.Metabolism variations of Ah strain induced by OXY were analyzed,which suggested potential relationship between stress and acquired tolerances.Key proteins related to OXY tolerance were selected and analyzed.In addition,preliminary studies were conducted to manage A.hydrophila using vaccine and efflux pump inhibitors.The key achievements of this tudy includes:1.Investigation on acquired resistance of A.hydrophilaOXY-Resistant strain(OXY-R)was induced from Ah after 10 generations under gradient increasing OXY.The MIC was increased from 2.5 ?g/mL to 40 ?g/mL.Comparison of the whole genomes of OXY-R and Ah detected multiple SNPs in outer-membrane-protein,antibiotic-resistant and virulence factor related genes,which indicates these genes are potentially attributed to the OXY-Resistance.Differential expression of outer membrane proteins was measured to investigate the difference between stress and acquired resistances using quantitative proteomics in OXY-R and Ah under the stress free conditions.The study suggested up regulated translation-related proteins and down regulated energy-metabolism related proteins in stress resistance.2.Investigation on stress resistance of A.hydrophilaComparison the proteomics of Ah under various OXY concentrations using MS meths.A total of 238 overlap proteins between iTRAQ and SWATH-MS were observed in the proteomics,In addition,translation-related proteins were unregulated under OXY stress,while the proteins related to the energy metabolism(glycolysis/gluconeogenesis and TCA cycle)were down-regulated.Outer membrane proteins of Ah were also investigated under various OXY concentrations using TMT coupled LC-MS/MS.A total of 259 overlap proteins two biological replicates were detected.Most of these proteins were related to ribosome process,oxidative phosphorylation process and ?-lactam resistance process.3.Comparison on stress and acquired resistances in A.hydrophilaIn comparison with stress and acquired resistances in A.hydrophila revealed that both mechanisms are up-regulated during translation and down-regulated during the energy metabolism process.Significant differences were found in virulence factors and outer membrane proteins,especially in the efflux pump system.The down-regulation of these two mechanisms during the energy metabolismwas further confirmed using NAD+and NADH analyses as well as activity assay of two TCA-cycle related enzymes.Toxic experiment showed the higher level of toxicity of OXY-R compared to Ah strain,which suggested the relationship between the virulence factors and drug resistances.q-PCR further confirmed that the differentiate expression of RND efflux pump proteins in strains of OXY-R and Ah under various OXY concentration.4.Study the biological function of the important proteins on OXY-resistance of A.hydrophilyFourteen candidate proteins were selected for further study including AcrA,LamB,MurA,SurA,BamE,MrcA,OprM,A0KJZ6,A0KN99 AOKHF7,AOKLQ6,AOKPL3,AOKR20 and AOKMB2.A total of 14 over-expression strains were generated through P9 expression system in E.coli.Nine of them showed significant difference(P<0.001)between control strains,which indicates the important roles of these proteins on OXY-Resistance.Among them,LamB and AOKLQ6 shows the most significant effect.LamB negatively regulates the OXY-Resistance,with the MIC value reduced 75%.AOKLQ6 positively regulates the OXY-Resistance,with the MIC value increased 3 times.The protein BLAST analysis indicates AOKLQ6 belonged to TolC family.5.Preliminary analysis on management of A.hydrophilyThis investigation suggests both virulence factors and RND efflux pump played an important role in the drug-resistance mechanism of A.hydrophila,which therefore can be used as specific targets for management.On the one hand,purified the recombinant proteins including AcrA,MurA,SurA,AOKLQ6,AOKHF7,A0KN99 and AOKPL3 were test as vaccine candidates.The challenge test showed that the relative protection rates(RPS)of zebrafish were more than 80%in AcrA,AOKLQ6?AOKBF7,A0KN99 and AOKPL3 while less than 10%in MurA and SurA.It suggest that the recombinant proteins including AcrA,AOKLQ6,AOKHF7,A0KN99 and AOKPL3 provide better protection efficacy in zebrafish against infectious caused by A.hydrophila and could be used to further anti-challenge test.One the other hand,reserpine,N-methyl-2-pyrrolidone and four Chinese herbology including astragalus,coptis chinensis,isatis root and fructus forsythia were investigated for their potential of A.hydrophila management.The in vitro antibacterial test and the resistance reversal test showed that reserpine has best effectiveness,followed by coptis chinensis.Western blotting analysis indicated the expression of the AcrA protein was inhibited by reserpine and subsequently affected the function of AcrAB efflux pump.This resulted in a reduction of excretion of OXY,which reversed the drug resistance. |
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