Preliminary Study On Microevolution Mechanism Of Resistance To Cefotaxime In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is an important foodborne pathogen,widely distributed in estuaries,marine environments and marine products.The growth temperature is 10⁴4 °C,and the optimum temperature is 35³7 °C.According to the official statistics of the National Foodborne Disease Surveillance System,V.parahaemolyticus is the main cause of foodborne bacterial poisoning in China,especially in coastal areas.Antibiotics are very useful as a drug for the treatment of human infectious diseases,and antibiotics have also been widely used in animal husbandry and aquaculture.The excessive use of antibiotics in aquaculture has led to an increase in Vibrio parahaemolyticus resistance.The application of genome-wide sequencing technology and transcriptomics technology can predict the potential drug resistance mechanism of microorganisms and further explore the reasons for bacterial resistance.In order to understand the mechanism of resistance evolution of V.parahaemolyticus,the following studies were carried out: 1.The resistance of V.parahaemolyticus to cefotaxime and the changes of drug resistance spectrum before and after evolution were induced.Phenotypic analysis of type and original V.parahaemolyticus;3.Obtaining drug-resistant and original genomics analysis of V.parahaemolyticus;4.Study on micro-evolution mechanism of cefotaxime resistance of V.parahaemolyticus.The specific content and results of this study are as follows:1.Artificial induction of resistance to cefotaxime by Vibrio parahaemolyticus and its changes in drug resistance spectrum before and after evolutionThis study established a method for inducing cefotaxime resistance in V.parahaemolyticus.The main steps are as follows: 1).Screening sensitive strains: Screening for cefotaxime-sensitive V.parahaemolyticus strains by broth dilution method.2).Sublethal concentration of cefotaxime for preliminary induction of V.parahaemolyticus: After treatment with the 0.5% MIC cefotaxime for the susceptible strain.3).Sublethal concentration of cefotaxime continuous induction: the broth was treated with 0.5 times MIC cefotaxime,and the strain was continuously grown under the sublethal cefotaxime concentration until V.parahaemolyticus The drug resistance of cefotaxime is produced,that is,the MIC value reaches the drug resistance standard prescribed by CLSI.V.parahaemolyticus was resistant to cefotaxime within 30 days,and the MIC was 512-fold from the original 0.125 ?g/m L to 64 ?g/m L on the 30 th day.On the 17 th day,VPC1 reached the drug resistance,reached the drug resistance level on the 28 th day,and the resistant strain was still resistant to cefotaxime after 8 passages,indicating that the resistance to cefotaxime obtained by VPC1 can be Stable inheritance.The strains induced after daily induction for 30 days were sequentially labeled as P0,P1,...,P30.Interestingly,P30 has developed resistance to the first-generation cephalosporin antibiotic cefazolin?KZ?and the second-generation cephalosporin cefoxitin?Fox?relative to P0?the original strain VPC1?,but to the third The cephalosporin ceftazidime?CAZ?remains sensitive.Flo R,sul I and sul II were detected in Vibrio parahaemolyticus P0 and P30,and there was no change in drug resistance genes before and after induction.2.Acquired drug-resistant and original transcriptomic analysis of V.parahaemolyticusFrom the perspective of transcriptomics,the preliminary mechanism of drug resistance evolution was revealed.The sensitive V.parahaemolyticus and the resistant strain of V.parahaemolyticus were sent to the BGI for transcriptome sequencing.The difference between the sensitive type of V.parahaemolyticus and the m RNA expressed by the drug-resistant V.parahaemolyticus was compared.There were 14 samples of planktonic and biofilm bacteria?three biological replicates?,corresponding labels.They are P0,P5,P10,P15,P20,P25,P30 and B0,B5,B10,B15,B20,B25,B30.The average output of each sample was 2.00 Gb,and the splicing and quality assessment was performed with reference to the genome of V.parahaemolyticus RIMD 2210633?NCBI accession: NC₀04603.1?,and the average alignment rate with the reference genome was 81.32%.According to the FPKM value of each gene,the gene correlation analysis between samples was carried out.The correlation coefficient between gene expression between P0 strain and P30 strain was 0.801,and the correlation coefficient between B0 and B30 was 0.796.The NOISeq method was used for screening differentially expressed genes in this experiment.The differential gene screening conditions were: q-value?corrected p-value?...