| Vibrio parahaemolyticus,a Gram-negative halophilic bacterium commonly distributed in water,aquatic animals or underwater sediments,is one of the main foodborne pathogenic microorganisms.The food safety incidents caused by it have increased year by year.In recent years,due to the extensive use of antibiotics in clinical treatment and fish farming,the resistance of V.parahaemolyticus has become increasingly serious,causing huge economic losses to the aquaculture industry.In the aquaculture process,quinolone antibiotics are commonly used in the treatment and prevention of vibriosis,so the resistance of V.parahaemolyticus to quinolones is also increasingly prominent.At present,there is still no continuous monitoring data on the epidemiology of V.parahaemolyticus resistance at home and abroad,and it is rare to study the resistance mechanism of V.parahaemolyticus to quinolones by detecting the expression of drug resistance genes in the continuous induction process by RT-q PCR.In this study,drug resistance of 30 strains of V.parahaemolyticus was tested by drug sensitivity test,and the carrying status of five quinolone resistance genes(including gyr A,par C,gyr B,par E and nor M)was detected by common PCR.And study on the acquisition rate and disappearance rate of ciprofloxacin and enrofloxacin resistance in the test strains,compared the MIC values of the strains before and after drug resistance induction.The rec A gene was selected as the internal reference gene,and the changes of m RNA expression levels of gyr A and par C genes in the drug resistance induction process was detected by RT-q PCR to used to study the molecular mechanism of V.parahaemolyticus resistance to quinolones.The main results of this experiment are:1.Thirty strains of V.parahaemolyticus were tested for drug susceptibility.The results showed that 30 strains of V.parahaemolyticus were highly resistant to sulfadiazine,sulfamethazine,sulfamethoxazole,methoxy pyrimidine and sulfadiazine,trimethoprim.The highest resistance rate was 100%,the highest susceptibility rate to flufenicol was 83.33%,followed by ofloxacin,the susceptibility rate was 80%.The sensitivity rates to enrofloxacin,norfloxacin,salafloxacin hydrochloride,doxycycline and neomycin were 40% ～ 70%.Multiple multidrug-resistant strains appeared,The dominant resistance spectrum was RAD/SD/SM2/SMM/SMD/SMZ,which accounted for 23.33% of the tested strains.The results of resistance gene detection showed that five quinolone resistance genes were detected in 29 bacteria except for the nor M resistance gene in strain 2017135.No gyr A gene mutation was detected.There were 1 to 3mutations in other drug resistance genes.The 75 mutation of par E gene may be related to low level drug resistance.The 99 mutation of nor M gene may be related to the mechanism of ciprofloxacin resistance.2.According to the results of the quinolone drug sensitivity test,3 strains(2014018,2015056,2017130)resistant to enrofloxacin and ciprofloxacin,and 3strains(2014135,2017129,2017137)were intervened and 3 sensitive strains(2013023,2013062,2017143)were randomly selected,the obtained rate test of resistance to ciprofloxacin and enrofloxacin showed that V.parahaemolyticus via ciprofloxacin and enrofloxacin induction of quinolone antibiotics,high drug resistance was obtained.Among them,strain 2013062 had the highest acquisition rate of resistance to ciprofloxacin,which was 256 times.The strains of 2014135,2017129,2017137 were also resistant to enrofloxacin.Acquisition rate is 256 times.In the study of the rate of drug resistance disappearance of the two quinolones,it was found that the resistance of the strain 2017143 to ciprofloxacin decrease 4 times,and the disappearance rate of ciprofloxacin in strains of 2014018,2015056,2017130 was 2 times,and the resistance of the remaining 5 strains to ciprofloxacin did not change;the resistance rates of enzyfloxacin in strains 2014135,2015056,2017129 and 2017143 were both 2times,and the resistance of other strains to enrofloxacin No change occurred,and the results showed that V.parahaemolyticus easily induced high-resistance mutants in low-concentration quinolones,and drug resistance was not easy to disappear within a certain period of time.3.Fluorescence quantitative PCR was used to detect the changes of par C resistance gene and gyr A resistance gene expression in each generation of tested strains(including strains 2013023,2013062,2017143,2014135,2017129,2917137,2014018,2015056,2017130)during induction of acquisition rate of ciprofloxacin and enrofloxacin resistance,and thelast generation of strains induced by the rate of disappearance of ciprofloxacin and enrofloxacin resistance.The internal reference gene selected in this experiment was rec A gene,and the data was analyzed by SPSS software.Graphpad Prism 5.0software was used for mapping.The experimental data showed that the expression of m RNA of V.parahaemolyticus par C and gyr A gene was proportional to drug resistance.The expression of gyr A gene and par C gene m RNA was detected by RT-q PCR for each generation of strains obtained at the rate of drug resistance and the last generation of drug resistance disappearance.It was found that V.parahaemolyticus was continuously induced in vitro by quinolone antibiotics.The expression levels of gyr A gene and par C gene showed an overall upward trend.In the induction of theresistance disappearrate of enrofloxacin and ciprofloxacin,the expression levels of gyr A gene and par C gene in the resistant group and the intermediate group were downe-rgulated in the last generation compared with the original one,and there were signifycant differences(P<0.05),The expression of the last generation of sensitive strains was significantly different from that of the original(P<0.01),indicating that the addition of the V.parahaemolyticus was resistant to quinolones is related to the expression of gyr A gene and par C gene,but the change of expression level during the induction process is not a continuous increase,which may be due to multiple drug resistance mechanisms or other regulatory mechanisms,which needs further study. |
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