| The issue of antimicrobial resistance has become one of the most pressing public health issues in the 21 st Century.As a major country in terms of R&D and antibiotic application,China is annually using about 162,000 tons of antibiotics for its medical heath and agricultural farming needs.The invention and the application of antibiotics have played a decisive role in curing infectious diseases to save patients' lives,preventing and curing animal epidemic diseases to enhance farming benefits,safeguarding safety in public health and other related fields over the past decades.However,the issue of antimicrobial resistance has become increasingly serious due to the fact that some drugstores do not have prescription to sell antibacterial agents;antibiotics are overused in clinical practice and farming;the effluent discharge of pharmaceutical industry is not up to the standards;and the weak awareness is prevalent among the public on proper drug application.In recent years,as a result of the lack of discovery linked to effective drug targets,new drug development has gradually slowed down worldwide.The emergence of more and more superbacteria indicates that mankind is gradually stepping into a “post-antibiotic era” and has even encountered a situation where no drugs are available for curing.The causes and consequences of antimicrobial resistance surpass the health field and have led to many adverse effects on human society,including more serious threat to biosafety,increased environmental pollution,and restricted economic development.Therefore,there is an urgent need to strengthen the control of the usage amount of antibiotic and improve the capacities of new drug development.Vibrio parahaemolyticus is a common gram negative marine halophilic bacteria.it can infect in fish,crustaceans,aquatic animal and is found in the gut flora of fish anf human.At present,in order to reduce the loss of aquaculture and medical field,the use of antibiotics is still the main method to control and treat the vibriosis.The overuse of antibiotics has caused the emergence of multi-drug resistant Vibrio parahaemolyticus,which has been paid more and more attention by experts and scholars all over the world.In recent years,studies have shown that exogenous addition of small molecule metabolites can increase the sensitivity of drug resistance of Edwardsiella tarda by aminoglycoside antibiotics.(1)In the present study,we aimed to explored the effect of adding three types of metabolites additional chemicals on enhancing drug susceptibility of Vibrio parahaemolyticus.The experimental results were conformed with carbonylcyanide-p-chlorophenyl hydrazine(CCCP).(2)By quantitative proteomics technology ITRAQ to compare the conditions in the presence of subinhibitory concentrations of antibiotics,exogenous addition of different kinds metabolites on the impact resistance of Vibrio parahaemolyticus in physiological state,in order to further explain the bacterial sensitivity to aminoglycoside antibiotics change mechanism.(3)At the transcriptional level,the quantitative expression of the differentially expressed proteins in proteomics was verified by RT-PCR.Through the analysis of the data indicated that exogenous glucose or alanine promotes by substrate activation,which in turn increases production of proton motive force(PMF)and primary sodium pump oxaloacetate decarboxylase and finally stimulates uptake of antibiotic.Therefore,we present a novel approach in fighting against multidrugresistant V.parahemolyticus with practical application value.1.Comparion of three types of metabolites on enhancing antimicrobial susceptibility of Vibrio parahaemolyticusAntibiotic resistance is a severe public health problem.Recently,the relationship between bacterial metabolic regulation and antibiotic efficacy has been proposed in several pathogens.In the present study,we explored the effect of adding additional metabolites on drug susceptibility of Vibrio parahaemolyticus.Antibiotic resistance has become an increasingly severe public health problem.Recently,the relationship between bacterial metabolic regulation and antibiotic efficacy has been proposed in several pathogens.In the present study,We aimed to explore the effect of three types of metabolites on enhancing drug susceptibility of Vibrio parahaemolyticus.We studied the phenotypic change and compared the percentage survival of V.parahemolyticus after treatment with antibiotics and different metabolites.The experimental results were conformed with carbonylcyanide-p-chlorophenyl hydrazone(CCCP).These results indicated that specific metabolites effectively potentiate kanamycin to eliminate multidrugresistant V.parahemolyticus and the significant bactericidal activity was only observed when treated with aminoglycoside in the presence of 10 mmol/L glucose.Treatment with CCCP was found to abolish metabolite-enabled potentiation by all of the carbon sources.The sensitivity of V.parahemolyticus to aminoglycoside can be improved by regulating the metabolism of bacterial cells.Furthermore,the mechanism of metabolite-enabled bacteria killing is dependent on the promotion of PMF generation and ultimately facilitates aminoglycosides uptake.Therefore,we present a novel approach in fighting against multidrugresistant V.parahemolyticus with practical application value.2.Proteomic analysis of three types of metabolites on enhancing antimicrobial susceptibility of Vibrio parahaemolyticus.In the present study,we untilized ITRAQ-based quantitative protemics to analyze the protein altertion in Vibrio parahaemolyticus 61 in response to subinhitory concentrations of Kanamycin in the presence of 10 mmol/L three types of metabolites.The results showed that a total of 178 protein spots with abundance changes in response to subinhitory concentrations of antibiotics.As reported,three types of metabolites subinhitory concentrations of Kanamycin by including stimulate central metabolic pathways and Na+ translocating decarboxylase is up-regulated.Superimposed on metabolic pathways related toaglucose,aalanineaandatheaTCAacycle,PDF(pyruvateadehydrogenase),Glt A(citrateasynt hase),ICDH(isocitrateadehydrogenas),OGD(oxoglutarataadehydrogenaseacomplex),SucC(succinyl-Co A synthetase)and SDH(succinate dehydrogenase)is up-regulated.Second,the expression levels of the key enzymes of the Aspartate and arginine biosynthesis pathway,Argininosuccinate lyase(argH),Argininosuccinate synthase(argG)was increased in the presence of metabolites.Interestingly,Na+-transporting oxaloacetate decarboxylase enzyme(OadGAB)was more aboundant after add exogenous metabolites in the presence of subinhitory concentrations of Kanamycin.Oad GAB,can restores the Na+ gradient and produces pyruvate.Na+ gradient may help coupling proton motive force and pyruvate to increase the uptake of the kanamycin.Here,we describe the metabolic state of antibiotic-resistant V.parahemolyticus and demonstrate that exogenous metabolites revert the bacterium to kanamycin susceptibility.3.Quantitative real-time PCR(qPCR)analysis for mRNA expression to verify differential proteins identified by ITRAQTo further verify the results of protein responses and compare the correlation between protein abundances and gene expressions,nine representative genes related to altered proteins were quantified using qRT-PCR technique.The results indicated that the levels of the gene expressions had same tendencies with corresponding proteins... |
PDF Full Text Request