Selection Of Multidrug Resistant Membrane-Associsted Proteins Of Streptococcus Suis Type2

In order to further find the molecular mechanism of antibacterial multidrug resistance of Streptococcus suis type2, in this study stepwise method was applied to induce3sensitive Streptococcus suis strains resistant to both erythromycin and ciprofloxacin in vitro, proteome method was used to select differential proteins related to resistance between sensitive and resistant Streptococcus suis strains, real time fluorescence quantitative RT-PCR was used to further confirm the mechanism which we have found.1Artificial induction of multidrug resistant mutants of Streptococcus suisA total of38clinical isolates of Streptococcus suis were examined for their susceptibility to6antibacterials. Stepwise method was applied to induce3sensitive Streptococcus suis strains resistant to both erythromycin and ciprofloxacin in vitro. The MICs of erythromycin and ciprofloxacin for each parent strain and its mutants were determined in the presence and absence of reserpine. PCR and gene sequencing were used to detect the resistance genes ermB、ermA、erm C、mefA、msrD、mphB、tetM、tetO、tetL、 tetK、23S rRNA、L4、L22and the quinolone resistance determining regions (QRDRs)of the DNA gyrase(parC and parE) and topoisomerase genes(gyrA and gyrB). The results showed that the rate of resistance to erythromycin and ciprofloxacin were39.5%(15/38)and28.9%(11/38) respectively. All clinical isolates of Streptococcus suis were resistant to sulfamonomethoxine and tetracycline, sensitive to ampicillin and florfenicol. Multidrug resistance mutants with significantly increased MICs (128mg·L-1) were generated from each parent strain. Isolates demonstrated no reduction in MIC in the presence of erythromycin with reserpine. Nevertheless, it revealed significant decrease of ciprofloxacin in the presence of reserpine, compared to that in the parent strain. The result of PCR and gene sequencing showed that all the multidrug resistance mutants were negative to ermB、 ermA、ermC、mefA、msrD、mphB, both tetM and tetO genes were detected. All the mutants had some changes in the quinolone resistance determining regions (QRDRs)of the DNA gyrase(parC and parE) and topoisomerase genes(gyrA and gyrB). 2Separation of differential proteins from sensitive strains to multidrug resistant mutants of Streptococcus suis type2by two-dimensional polyacrylamide gel electrophoresisSensitive strains to multidrug resistant mutants of Streptococcus suis were cultured, and the membrane-associated proteins (MAP) were extracted from these baeteria by Triton X-114extraction. After preparation of the MAP, solubilized proteins separated in the first molecular in IPG(immobilized pH gradient)strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis. Those differential protein spots were visualized by image analysis software of ImageMaster5.0. The differential protein spots were excised and analyzed with MALDI-TOF-MS. The results showed that the resolution of gel electrophoresis were high. It was found that there were (311±53) protein spots in protein2-DE gels of sensitive strain,and (237±53) protein spots in that of multidrug resistance mutants.9proteins related to multidrug resistance were identified based on peptide mass fingerprinting the results indexed in database. The function of these identified proteins were related to metabolism, virulence and resistance of Streptococcus suis.3Investigation of mRNA expression of differential proteins related to resistance by Real time RT-PCRIn order to make clear whether the ABC transporter played a part in Streptococcus suis multidrug resistance, real time fluorescence quantitative RT-PCR was used to investigate the mRNA expression of gene SS2069corresponding to ABC transporter in sensitive strain JR05730, Intermediate strain JR05730-M and resistant strain JR05730-EC of Streptococcus suis. The results showed that the mRNA expression of gene SS2069of intermediate and resistant strain increased4.7and6.3folds respectively with the resistance of Streptococcus suis increasing. This result further evidenced that the ABC transporter protein was related to the resistance of Streptococcus suis.