| Vibrio parahemolyticus is one of the important zoonotic pathogens and mainly derived from seawater and marine products,which caused typical gastroenteritis symptoms,such as diarrhea,nausea,fever and so on.The pathogenesis of the disease has not been fully clarified,but the thermostable direct hemolysin(TDH)and thermostable related hemolysin(TRH)are now recognized as the major virulence factors of Vibrio parahaemolyticus.In recent years,the irregular usage of antibiotics in aquaculture and clinical practice has led to the increasingly serious drug resistance of Vibrio parahemolyticus and caused great economic losses to the aquaculture industry.Study on the lack of continuous monitoring data of the epidemiology of drug resistance at home and abroad,and less research in the mechanism of drug resistance.In this study,we investigated the distribution and drug resistance of 107 Vibrio parahemolyticus strains from different sources,furthermore,the model of antibiotic resistant strains was induced by the antibiotic gradient concentration in vitro,study on the mechanism of resistance of quinolones by mutation analysis.The main research methods and results are as follows:(1)Identification of 107 Vibrio parahemolyticus strains is finished by Compact VITEK-2,the virulence-related genes were screened by PCR amplification,the tlh gene was detected in all isolates,the positive rates of tdh,trh and orf8 were 18.7%(20/107),8.4%(9/107)and 15%(16/107),while all orf8-positive isolates carried tdh gene.The positive rate of virulence gene of clinical isolates was much higher than that of environmental isolates,which indicated that there were potentially pathogenic strains in clinical isolates.The PCR results of VPA0166(encoding putative outer membrane protein gene)among 107 isolates showed that there were 55 isolates showed type-A(996bp)and 52 showed type-B(1050bp).There were 54 base deletions between 523bp and 580bp in type-A products.Compared with the corresponding sequences of Vibrio parahemolyticus RIMD 2210633,the sequence homology was 94%with type-B products,it showed that the type-B strains were closer to the sequenced strain in the course of evolution.The diversity of VPA0166 has correlated with tdh and trh genes carrying,all tdh+ strains belong to type-B,and trh+ strains belong to type-A.(2)The results of antimicrobial resistance showed that resistance strains were found to Cefoxitin,Tetracycline,Sulfonamides.The rate of resistance to cefoxitin was 1.9%(2/107),there was a strain of Tetracycline,Sulfonamides,which genotype was tdh-trh-orf8-,the medium sensitive rates to cefoxitin and ciprofloxacin were 94.4%and 44.9%.The medium sensitive rates to Ciprofloxacin of strains with tdh+trh-/tdh-trh+ was 10 percentage points higher than with tdh-trh-,inferring potential pathogenic Vibrio parahaemolyticus strains have better tolerance than nothing.The medium sensitive rates to ciprofloxacin of VPA0166 type-B strains were significantly higher than type-A strains(P<0.05),it indicated that type-B strains are more resistant to ciprofloxacin,which are more likely to develop into dominant bacteria under long-term antibiotics.(3)Grouping with different antibiotic resistance and VPA0166 genotype,amplification of chromosome mediated quinolone resistance gene in 40 isolates using PCR method,it was found that the low level of resistance related to mutation of 75 site in the parE gene,the 96 and 117 sites mutations in multidrug efflux pump NorM had connection with mechanisms of resistance to ciprofloxacin.In addition,detection of plasmid mediated quinolone resistant(PMQR)genes of all isolates showed that a total of 10 PMQR-positive strains were detected,and the positive rate was 9.3%(10/107).The gene qnrC,qnrS,qnrVC existed in 5.6%(6/107),0.9%(1/107)and 2.8%(3/107)respectively,there were base mutations.To establish a stable and reliable model of resistant strains by continuous subculture in the ciprofloxacin gradient medium,The MIC increased by 64～128 times.Detection of QRDR and efflux pump genes showed that the gyrB,parE had no mutation,Ser83→Ile mutation of gyrA gene were mostly occurred which widely existed in highly resistant strains,gyrA is the main target gene.Nucleotide mutation occurred at 369 site of when MIC of ciprofloxacin was 16 μg/mL,while the corresponding amino acid sequence did not change.There were mutation of 83 site in the gyrA gene of VP1276R,moreover the Ser85→Phe mutation of parC was occurred.The results of in vitro experiments on Vibrio parahaemolyticus strains for induced resistance to ciprofloxacin showed that the presence of gyrA and parC gene mutation strains belong to VPA0166 type-B,inferring that VPA0166 type-B strains are more prone to mutation of antibiotic resistance gene by long-time application of antibiotic,which led to the emergence of antibiotics resistance. |
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