Preliminary Study On The Mechanisms Of β-lactam Antibiotic Resistance In Vibrio Parahaemolyticus And Vibrio Anguillarum

Vibrio parahaemolyticus and Vibrio anguillarum are two important pathogens of fishery diseases.Vibriosis caused by them is one of the most common fish diseases,leading to huge economic losses to fishery production.Many studies have focused on the pathogenic mechanisms and virulence factors of Vibrio parahaemolyticus and Vibrio anguillarum and shown that Vibrio parahaemolyticus and Vibrio anguillarum exhibited multi-drug resistance.The resistance to β-lactam antibiotics of Vibrio parahaemolyticus and Vibrio anguillarum was discovered in our previous studies.,but with different extents.At present,there are many studies on the mechanisms of antibiotic resistance of Vibrio parahaemolyticus,especially the effect on β-lactamase,but there are few researches on the mechanisms of antibiotic resistance of Vibrio anguillarum.In this paper,the pathogenic potential of Vibrio parahaemolyticus D3112 isolated from the seaside was mainly examined,and its pathogenic genes and drug resistance genes were analyzed by genome sequencing.Besides,we analyzed the antiampicillin resistance of Vibrio parahaemolyticus D3112 and Vibrio anguillarum 775.The results provide guidance for preventing the prevalence of Vibrio and views on the mechanism of resistance to Vibrio.The main contents of this paper are as follows: 1.The role of β-lactamase in Vibrio parahaemolyticus D3112A strain of Vibrio parahaemolyticus D3112 was screened by traditional separation method and its whole genome was sequenced.The virulence genes and drug resistance genes were analyzed.Two β-lactamase genes lac1 and lac2 in Vibrio parahaemolyticus D3112 were identified by genome sequence analysis.In vitro enzyme activity experiments showed that Lac1 protein and Lac2 protein could degrade ampicillin.Two single deletion mutants were obtained by gene knockout,and the differences in drug resistance between wild type and the mutant strains were compared.The results showed that the β-lactamase encoded by the lac2 gene plays a major role in D3112,while the deletion of the lac1 gene has no significant difference with wild type on drug resistance.2.Study on drug resistance of Vibrio anguillarum 775 RND efflux pumpFive RND efflux pump operons vaeAB,vaeCD,vaeEF,vaeGH and vaeIJ in Vibrio anguillarum 775 were found by bioinformatics.The RND efflux pump single deletion mutant strains were obtained by gene knockout,and the difference in drug resistance spectrum between wild type and each mutant strain was analyzed.It was found that the VaeAB efflux pump played a major role in the β-lactamase antibiotic resistance of Vibrio anguillarum 775.The expression of vaeB mRNA was detected by qRT-PCR under different concentrations of ampicillin,the results indicated that ampicillin can up-regulate the expression of the vaeB gene.Through the EB aggregation experiment,it is explored that the VaeB pump is powered by the proton pump.The biological analysis and experimental verification proved that the VaeB pump is an RND drug efflux pump.