Study On The Relationship Between Fluoroquinolone Sensitivity And Gene Mutations Of Streptococcus Suis

Streptococcus suis infection is a kind of polytypism plague caused by Streptococcus suis which is now one of the most important factor on pig husbandry loss. SerotypeⅡhas been discovered to be the most frequently associated with zoonosis. Meningitis and septicemia of human can also be caused by S.suis. It is crucial and inevitable for pig industry of many countries about how to prevent and cure S.suis infection effectively. According to recent reports, isolations of S.suis strain resistant to fluoroquinolones is getting commonplace.The action mechanism of fluoroquinolones is to inhibit the activity of topoisomeraseⅡand topoismeraseⅣby creating a Quinolone-DNA-topoisomerase complex so that bacteria might die because its normal cleavage can not be completed. Resistance mechanism reported about S.suis against fluoroquinolones is mutations of target site and efflux pump. TopoisomeraseⅣis made of two subunit: ParC and ParE, encoded by parC and parE. DNA gyrase which catalyzes ATP-dependent DNA supercoiling is made of GyrA and GyrB, encoded by gyrA and gyrB gene. With GyrA and ParC subunits of resistant bacteria, amino acid changes are generally localized to a region of the enzyme in the amino terminus that contains the active site that is the mutation sites which are often found in a certain stegnotic segment between residue 67 and 106 of ParC and GyrA, named QRDR of the two genes. There isn't any report about the relationship between S.suis resistance to fluoroquinolones and the mutation of parC and gyrA genes in China until now. This experiment will study on the mutation of the two genes of S.suis isolated from the Northeast of China in order to provide useful basic scientific information for further research on S.suis and the proventation of S.suis disease.Srains of S.suis were isolated from hogpens of northeast area using nasal swabs. 100 srains were identified after biochemical reaction and lancefield group experiments. Pathogenicity was tested on Kunming mice. Resistant strains against 9 kinds of common used antibiotics and 11 kinds of fluoroquinolones drugs were selected by disk susceptibility test and microdilution susceptibility test. The mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes from 28 strains of resistant bacteria were analyzed by polymerase chain reaction (PCR), the production of PCR was purified and sequenced and analyzed. The results of biochemical reaction are like these: all strains didn't live in 6.5% NaCl broth and hippurate, 57% strains did not live in the condition of raffinose or alantin; 98% strains could make use of manicol and lactose; 90% of strains decomposed sorbitol; all strains could live with esculin, salicin, Trehalose; 98% strains are identified as D type with the test of lancefield group. The results of antibiotic sensibility against nine common drugs are like these: 69% strains were resistant to alficetin, and 75% to cidomycin, 96% to erythromycin, 82% to penicilin G, 91% to trimethoprim- sulfamethoxazole, 98% to ampicillin, 100% to oxacillin, 97% to tetracycline, 48% to ciprofloxacin. The results of antibiotic sensitivity tests of S.suis against 11 kinds of fluoroquinolones are like these: 48% strains were resistant to ciprofloxacin, 45% to gatifloxacin, 51% to sarafloxacin, 34% to levofloxacin, 85% to lomefloxacin, 30% to ofloxacin, 86% to fleroxacin, 69% to norfloxacin, 35% to enrofloxacin, 53% to difloxacin, 75% to pefloxacin;Homology analysis indicated that parC and gyrA genes of 28 strains have the homology of 89.3% and 91.6% respectively, and the homology with those standard srains submitted online were 74% and 80% respectively. Nucleotide mutaion mostly occurred among the 33~264 sites of parC genes and 57~369 sites of gyrA genes. S80→I mutation of parC gene were occurred in 18strains of fluoroquinolone resistant S.suis and Ser81→Ior F or Y of gyrA gene in 11 highly resistance strains; no amino acid change in gyrA or parC was detected when strains MIC of ciprofloxacin were under 1μg/ml.A single substitution in parC was found in the strains whose MIC of ciprofloxacin was higher than 2μg/ml.Strains in which the mutation of S81 of gyrA were occurred at the same time had a high resistance level. Mutations in parC gene result in low level resistance against fluoroquinolones, and high level resistance is the outcome of the mutations in both genes.The numbers of mutations is proportional to the level of fluoroquinolone resistance.