| Vibrio parahaemolyticus is a kind of halophilic Gram-negative bacteria,which widely exists in fishery environment,fish,shellfish,shrimp and other common seafood.Vibrio parahaemolyticus can affect the growth and development of organisms,such as shrimp suffering from acute hepatopancreatic necrosis disease(AHPND).Vibrio parahaemolyticus is transmitted through the food chain of aquatic products,which poses a great threat to human health.People infected with Vibrio parahaemolyticus will have diarrhea,headache,nausea,vomiting,abdominal spasm and other reactions.Since Vibrio parahaemolyticus is a potential threat to human health and economic development,it is imperative to control its reproduction in fishery production.In general,antibiotics are used to control Vibrio parahaemolyticus.In recent years,it has been found that the antibiotics resistance of Vibrio parahaemolyticus strains isolated from aquatic products,fishery environment shows an increasing trend,and the existing strains show multiple drug resistance.In addition,virulence genes and drug resistance of Vibrio parahaemolyticus from different regions are significantly different,which makes prevention and treatment difficult.Therefore,the rapid identification of drug resistance of Vibrio parahaemolyticus is of great significance for the scientific use of antibiotics in fishery production.The antimicrobial susceptibility testing is the most effective tool for the determination of microbial resistance.Among them,the phenotypic methods(such as dilution method,disk diffusion method,e-test method,turbidity method,etc.)based on the determination of the effect of target antibiotics on microbial growth activity are still the gold standard.However,the existing methods,especially manual methods,have some problems such as low efficiency and poor accuracy.Although the automated method based on turbidity analysis has relatively high accuracy,it is inefficient because of the tedious pretreatment steps.Therefore,the high efficiency,easy to operate,low cost AST method is still an urgent need of the industry.In order to determine the antibiotics resistance of Vibrio parahaemolyticus,a new antimicrobial susceptibility testing method based on microbial growth sensor was developed.There are two main contents:one is the establishment and performance characterization of the new automated and high-throughput method,and the other is the practical application of the method in the determination of resistance of environmental isolates.(1)Using capacitively coupled contactless conductivity detection as the sensitive element,an eight-channel microbial growth sensor AST method was established.With the standard strain of Vibrio parahaemolyticus(ATCC 17802)as the research object,the sensor can be used to monitor the changes of electrical conductivity caused by cell metabolism in each working channel in a non-destructive and online manner,so as to automatically form a dynamic growth curve.Eight-channel microbial growth sensor was used to monitor the growth curve of 5×10~5CFU/m L Vibrio parahaemolyticus(ATCC17802)in the presence of antibiotics in real time,and the microbial growth sensor AST method was established.The results showed that Minimal Inhibitory Concentration(MIC)of kanamycin,tetracycline,penicillin and enoxacin were 10.0,2.0,1280 and 0.125?g/m L in LB liquid medium for 20 h,respectively.Essential Agreement(EA)of results between microbial growth sensor and broth microdilution method was 100%,the reliability of the new method is verified.(2)Sixteen strains of Vibrio parahaemolyticus were isolated and identified from fishery environment and aquatic products,and their drug resistance in LB liquid medium was determined by broth microdilution method.The results showed that the MIC value of kanamycin was between 2.5 and 20.0?g/m L when cultured at 35?for 20 h.The MIC value of tetracycline ranged from 1.0 to 32.0?g/m L.The MIC value of penicillin ranged from 40 to 640?g/m L.The MIC value of enoxacin is between 0.03125 to 16.00000?g/m L.These data indicate that there are significant differences in antibiotic resistance/sensitivity of isolated strains.There was no difference of more than three times concentration gradient in the broth microdilution method control experiment,and it was basically no higher than the data obtained by the AST method of microbial growth sensor,indicating that the newly developed automated method have higher sensitivity.In this study,we established a new method for the determination of antibiotics resistance of Vibrio parahaemolyticus based on a sensor model that can online nondestructively monitor the microbial growth process.Compared with the common methods,the new method has the advantages of simple operation,high efficiency and low cost,which lays a foundation for the construction of more antimicrobial sensitivity testing platforms.The results of antimicrobial sensitivity testing of isolated strains from environment and aquatic products provide reliable data for guiding scientific antibiotics use in fishery production. |
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