| Multidrug resistance of Streptococcus suis have been spread in worldwide. Both mutations within the QRDRs and efflux pump contribute to the resistance to fluoquinolones. This study aims to explore the mechanism involved in fluoquinolones resistance in Streptococcus suis.Firstly, total of four clinical susceptibile isolates and nine induced resistant strains of Streptococcus suis were examined for their susceptibility to enrofloxacin, ciprofloxacin, ofloxacin and danofloxacin and the growth curve of susceptible and resistant strains were tested. PCR was used to amplify the target genes and sequencing was done to detect the mutation of QRDRs of gyr A, gyrB, parC and parE. Then, reserpine was combined to detect the MICs of enrofloxacin and ciprofloxacin against induced Streptococcus suis and growth inhibition assays as well as accumulation of ciprofloxacin in susceptibile and resistant strains were performed at the same time. To further investigate the correlation of SatA, SatB and SmrA in contributing to fluoroquinolone resistance in Streptococcus suis, Real time RT-PCR was used to compare the different expression level of sat A, satB and smrA in resistant and susceptible strains. At last, we tried to use mutagenetic method to get satB mutant to find out the contribution of satB to FQs resistance.Results showed that all induced Streptococcus suis mutants had been resistant to fluoquinolones. However, growth curves showed no significant difference between susceptible and resistant strains, which indicating that resistant strains could grow well in resistant strains, there existed some amino acid substitions within QRDRs of gyr A, gyrB, parC and parE. Most of the mutations were Ser81Arg in gyr A and Ser79Phe in parC, which were accordant with previously reported. However, there were some mutations which had not been reported everywhere. Most interestingly, there were no mutations in QRDRs of gyr A, gyrB, parC and parE of some FQs-resistant Streptococcus suis strains. This suggested other mechanisms may involve in the resistance to fluoquinolones in S. suis.Further results showed that, when reserpine was added, most resistant strains restored sensitivity to ciprofloxacin with the MIC values decreased by2-16folds. Reserpine could inhibit the growth of resistant strains in the presence of1/4><MIC ciprofloxacin, which suggested an efflux pump drived by energy released through hydrolyzing ATP. The results of accumulation showed that resistant strains can pump out ciprofloxacin easier than that of susceptibile strains. When reserpine was added, resistant strains could uptake more ciprofloxacin, which further confirmed an efflux pump system was involved in FQs resistance in Streptococcus suis.Results of Real time RT-PCR showed that the expression level of satA and satB is significantly higher in strains JS12B and JS13B, which had no site mutation than that of their parental susceptible strains. While, in strains JS14B and JS18B, which had mutant points, the expression level of satA and satB were increseased, but not as high as in JS12B and JS13B. The expression level in strains induced by enrfloxacin was relatively lower than strains induced by ciprofloxacin. The expression level of satA and satB was respectively1.1-2.0and1.1-2.4fold higher in strains induced by ciprofloxacin at the concentration of1/4xMIC respectively. The expression level of satA and satB in JS14E strain was decreased by2.5and2.1fold respectively, when1/4×MIC concentration ciprofloxacin was added. While expression level of satA, satB of the JS18E2strain was respectively increased by1.76and1.64fold in strains induced by enrofloxacin when1/4×MIC ciprofloxacin was added. While the expression level of smrA displayed no significant change in susceptibile parental strains and induced resistant strains.In order to further study the mechanism of satA and satB in mediating the resistance to fluoroquinolones, a thermosensitive suicide vector pSET4s was used to construct the AsatB plasmids, pSET4s-L-Cat-R and pSET4s-RH, respectively, using chaoramphenicol substitution and fusion-PCR method. Then these plasmids were electro-transformed into Streptococcus suis strain JS12B, respectively. However, no positive site mutanted strain was found in our selection. It implied that satB may be important for the growth and metabolism of Streptococcus suis. Strains with satB knocked out can not survive. Other mutagenetic method should be taken to investigate the satA and satB function in mediating ciprofloxacin resistance in S. suis. Above results demonstrate that a double-efflux pump is correlated with ciprofloxacin resistance in S. suis. |
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