| Vibrio parahaemolyticus(Vp),is a Gram-negative halophilic bacteria,widely present in aquatic products.It was one of the main food-borne pathogenic microorganisms which caused food safety incidents increased every year.With the widespread use and abuse of antibiotics in clinical treatment and aquaculture,Vibrio parahaemolyticus had become more resistant to antibiotics.Quinolone antibiotics have been widely used in the treatment and prevention of Vibrioosis,resistance of quinolone antibiotics for Vibrio parahaemolyticus was increasingly significant.At present,there have a little researchs of the mechanism of quinolone antibiotics resistance in Vibrio parahaemolyticus.In this study,the antibiotic susceptibility,growth curve,quinolone resistance-determining region sequence of gyrA and parC genes,and the expression rate of RND-type efflux pump were evaluated in induced strain and the parent strain,along with the analysis of whole genome sequence of parental strain,the molecular mechanism of Vibrio parahaemolyticus to quinolones has been preliminary analyzed.The main results were as follows:(1)Changes of resistance phenotype before and after in vitro inductionThe four resistant strains were obtained from in vitro induction of F7 with ciprofloxa cin,ampicillin,tetracycline,?-polylysine and tea polyphenols,respectively.It was found that antibiotics were more likely to induce Vibrio parahaemolyticus becoming multiple antibiotic-resistant.For example,for Ciprofloxacin-induced strain H128,the Norfloxacin and ciprofloxacin MIC increased by 1000 times;Ampicillin-induced strain A128 increased the MIC of nalidixic acid,ciprofloxacin,norfloxacin and cefazolin by more than 100 times;Tetracycline-induced strain S128 increased the MIC of tetracycline by 64 times.?-polylysine-induced strain ?8,only presented resistance to tetracycline,cefazolin and amoxicillin.The adaptive analysis showed that the logarithmic growth phase of the resistant strain was prolonged,but the final bacterial concentration could reach or exceed that of the parental strain.The resistance to antibiotics of H32 and H128 induced by ciprofloxacin was relatively stable,however,the resistant appears to recover in ampicillin-induced strain A128 after passage.(2)The genome sequencing of the parental strain F7Using the single molecule PacBio sequencing,and a variety of software analysis,the genes function was annotated.Through Phylogeny tree analysis,It was found that the isolate F7 was closest to the two clinical strains.Analysis of resistance genes showed that there were 64 genes of the efflux pump,8 tetracycline efflux genes,5 ?-lactamase genes,1 chloramphenicol resistance gene,1 quinolone antibiotic resistance gene.The homology analysis of RND type efflux pump genes showed that,26 homologous genes were present in F7 withmore than 95% similarity with the reference sequences.(3)The quinolones-resistance mechanism study of Vibrio parahaemolyticusThe QRDR of the gyrA and parC genes in resistant strains and the parental strain F7 was amplified by PCR and sequenced,and compared with the reference sequences.It was found that only gyrA gene of H32,H128 occurred Ser83-Ile mutation,the parC gene of all strains did n't occur non-synonymous mutation.The expression levels of vmeB,vmeD and vpoC were measured by reverse transcription-polymerase chain reaction(PCR).It was found that the expression levels of vmeD and vpoC were 9.71 and 3.43 times higher than those of the parental strain F7.In the H32,S32 and S128,the vmeD gene was up-regulated by 1.5-2.1 times,and the vmeB gene was down-regulated in all the resistant strains.Thus,it was indicated that the VmeCD-VpoC was the main efflux in H128. |
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