| Vibrio parahaemolyticus is one of the common foodborne pathogenic bacteria,and which is the leading cause of food poisoning events throught the world.Vibrio parahaemolyticus has been regarded as one of the main cause of the outbreak of diarrheal disease in the world.The antibiotic resistance of Vibrio parahaemolyticus has attracted much attention.However,the reasons for the microevolution mechnism of antimicrobial resistance of Vibrio parahaemolyticus have not been explained in details.The application of whole genome sequencing technology will help to monitor the emergence and transmission of antibiotic resistance,to predict the potential antimicrobial resistance mechanism,and further to explore the causes of acquire bacterial resistance.This technology was play an important role in the diagnosis,treatment and the research of new drugs.Therefore,in order to solve the microevolution of antimicrobial resistance of Vibrio parahaemolyticus,some researches has been done as follows:(1)A comparison of antimicrobial resistance of pathogenic Vibrio parahaemolyticus isolated from seafood and clinical samples;(2)An induced method of levofloxacin resistance of Vibrio parahaemolyticus;(3)Research on the changes of the biological traits of the sensitive and drug-resistant Vibrio parahaemolyticus;(4)Tracking a microevolution mechanisms of antimicrobial resistance of Vibrio parahaemolyticus with Whole genome sequencing.(1)A comparison of antimicrobial resistance of pathogenic Vibrio parahaemolyticus isolated from seafood and clinical samplesThe aim of this study was to reveal the difference of antimicrobial resistance among the pathogenic V.parahaemolyticus isolates from seafood and clinical samples,which provides the data basis for the antibiotic resistance mechanism of V.parahaemolyticus.A total of 46 pathogenic V.parahaemolyticus were isolated from clinical samples(n=28)and seafood samples(n=18).Antimicrobial resistance phenotypes(18 antimicrobials;disk diffusion method)and genotypes(38 antimicrobial resistance genes;PCR)of 46 pathogenic V.parahaemolyticus were evaluated.Furthermore,the genetic diversity of 46 isolates was analyzed by multilocus sequence typing(MLST),and the antimicrobial resistance of isolates in the same clonal complexes was compared.The results showed that the antimicrobial resistance rate of 28 clinical isolates(100%)was obviously higher than that of 18 seafood-derived isolates(88.9%).And all clinical isolates were multi-drug resistance bacteria(n = 28),while only 2 seafood-derived isolates could tolerate more than 3 kinds of antimicrobials.Additionally,the number and kind of antimicrobial resistance genes of the clinical isolates were more complex than seafood-derived isolates.The similar phenomenon was also observed in these isolates which belonged to the same clonal complexes.This study compared the difference of antimicrobial resistance between seafood-derived and clinical V.parahaemolyticus isolates,and primarily analyzed microevolution of antimicrobial resistance in V.parahaemolyticus.This study could provide the scientific support for study of the origin,spread and control of the antimicrobial resistant V.parahaemolyticus.(2)An induced method of levofloxacin resistance of Vibrio parahaemolyticusIn this research,we developed a method to rapid inducing of Vibrio parahaemolyticus to acquired levofloxacin resistance.The main steps have been done as follows.Firstly,is screening of sensitive Vibrio parahaemolyticus strains.The minimum inhibitory concentration(MIC)of levofloxacin sensitive isolates were determined by a multiple dilution method of broth,and we selected the strains of levofloxacin sensitive of Vibrio parahaemolyticus.Secondly,the step is preliminary induction of sublethal antibiotic concentration.The treatment of sensitive bacteria strains can grow in 1/2MIC.And this isolates were cultured in different concentrations of antibiotics in 96 well plates.Thirdly,the procedure is continuous induction of sublethal antibiotic concentration.During the process of repeating this method,we acquired the levofloxacin resistance Vibrio parahaemolyticus,and which is growed in the sublethal antibiotic concentration.Its MIC value reaches the standard of drug resistance prescribed of CLSI.The fourth step is keep in store the antimicrobial resistance isolates.We collected the strains that was cultured after induction to 9 Log CFU/mL.The last step is to verified the drug resistance of Vibrio parahaemolyticus.The method is that we reactivated and continuously cultured the mutation strains for 5 days.And two different drug sensitivity tests were used to detect the resistance of the mutation strain.The results showed that the induced Vibrio parahaemolyticus acquired the resistance of levofloxacin.Simultaneously,this resistance can be inherited steadily.In this study,a method of inducing levofloxacin resistance in Vibrio parahaemolyticus was constructed.It can quickly obtain the antimicrobial resistance isolates with stable genetic characteristics.It provides a reliable tool for the study of the mechanism of levofloxacin resistance to Vibrio parahaemolyticus.Meanwhile,it has good scientific research value and practical significance.(3)Research on the changes of the biological traits of the sensitive and drug-resistant Vibrio parahaemolyticusIn order to study the changes of the biological traits of the sensitive and drugresistant Vibrio parahaemolyticus,the antimicrobial resistance,microstructure,growth,exercise,and biofilm of the sensitive VPD14 and the resistant VPD14 M were investigated,respectively.The results showed that the levofloxacin resistance isolate VPD14 M was also resistant to the same antibiotic to ciprofloxacin,and it lost the resistance of aminoglycoside antibiotics at the same time.Meanwhile,there was no significant difference in microstructure between sensitive strain VPD14 and drug resistant strain VPD14 M,but the growth ability and motility of sensitive strain were stronger than those of resistant isolate.Furthermore,the analysis of the ability of biofilm formation of sensitive strain VPD14 and drug resistant strain VPD14 M indicated that biofilm forming capability of VPD14 M was significantly higher(p < 0.05)than that of VPD14.The amount of biofilm and the thickness of biofilm of drug resistant strain VPD14 M were also significantly higher than sensitive isolate.This research comprehensive and systematic analysis the changes of biological traits of the sensitive and drug-resistant Vibrio parahaemolyticus,and provided the basic data for research the microevolution mechanism of antimicrobial resistance of Vibrio parahaemolyticus.(4)Tracking a microevolution mechanism of antimicrobial resistance of Vibrio parahaemolyticus with Whole genome sequencingIn order to explore the causes of antimicrobial resistance of Vibrio parahaemolyticus,further to reveal the microevolution mechanism of antimicrobial resistance.Based on the whole genome sequencing technology,this research compared the genomic differences between the sensitive isolate VPD14 and the acquired strain VPD14 M by the second generation sequencing technology and the third generation sequencing technology.Besides,we analysied the identity of antimicrobial resistance of the sensitive and the drug resistance Vibrio parahaemolyticus.The results showed that the whole genome of sensitive isolate VPD14 is 1,282,695,483 bp with the average GC content is 42.84%.The sensitive isolate VPD14 genome harbors two circular chromosomes of 3,291,121 bp and 1,877,519 bp,the average GC content is 45.38% and 45.36%,respectively.The whole genome of antimicrobial resistance Vibrio parahaemolyticus is 5,089,287 bp with the average GC content is 45.29%.Through the comparative genome,50 and 48 resistance-related genes were identified in the sensitive and drug-resistant isolates,respectively.Meanwhile,91.7% of the gene sequences were identical in all the same resistance-related genes.What makes the results especially interesting is that only gyrA,parC and gyrB have a single base mutation.And the mutations of these three quinolones belong to the non synonymous mutation.Therefore,in this research,we preliminarily revealed the microevolution potential mechanism of antimicrobial resistance of Vibrio parahaemolyticus by the comparative genomics techonogy.Under the stimulation of sublethal concentration of antibiotics,Vibrio parahaemolyticus is adapted to the environment,its resistance gene gyrA,parC,gyrB were all mutated.We revealed that it caused the production of corresponding quinolone antibiotic resistance. |
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