| Streptococcus suis is one of bacterial infectious diseases on pig husbandry in today’s world. The most common pathogenic serotype is type2, which can cause such as septicemia, endocarditis, meningitis, septic arthritis, pneumonia and abortion of pigs. The germ can cause septicemia, endocarditis and meningitis, even death in case of seriousness if it infects human beings. The fluoroquinolones are a class of broad-spectrum, potent synthetic drugs of chemosynthesis; it has been widely used for a variety of bacterial infection in clinical practice, which is considered to be effective therapy for Streptococcus suis. However, its drug resistance will be generated and gradually increased when the drug has been widely long-term used and unreasonable applied in human medicine and veterinary clinically.The resistance mechanism of Streptococcus suis serotype2(SS2) to fluoroquinolones is rarely reported. In order to clarify the mechanism resistance of SS2to fluoroquinolones and provide theoretical basis for the prevention and treatment of SS2, this study was performed to make drug resistance inducing of norfloxacin and enrofloxacin for four strains SS2that are sensitive to norfloxacin and enrofloxacin isolated from the region of Guangxi and Guangdong. The MIC of norfloxacin and enrofloxacin was increased to256μg/mL by resistance induction in vitro. The study clarified the mechanism of the SS2resisting to fluoroquinolones. By studying the parental strains and their respective induced to a high degree of drug-resistant strains gyrA, parC genes of fluoroquinolones resistance determining region (QRDR) the mutation of amino acid, detection drug-resistant strains of efflux pumps, drug-resistant strains of resistant plasmids to detect and eliminate, whether the resistance plasmid conjugal transfer into E. coli, resistance to different fluoroquinolones Streptococcus suis serotype2is a cross-resistance and other aspects in order to clarify the mechanism resistance of SS2to fluoroquinolones.Biochemical tests and PCR methods identified six SS2strains. The results showed that six strains of biochemical tests:all strains didn’t live in40%bile salts, methylene blue milk,6.5% sodium chloride, gelatin liquefaction and hippuric acid I,67%strains didn’t survive in sorbitol and inulin, all strains can live with starch, mushrooms, sugar and glucose,83%strains make use of raffinose and aescin glycosides,67%strains decomposed sorbitol lactose,50%strains can live in starch. Six of SS2was amplified by PCR from15clinical isolates of Streptococcus suis strains, The isolation rate of40%of the separation of the total bacterial counts, including Guangxi5, Guangdong1. The MIC of enrofloxacin and norfloxacin against six SS2strains MIC were determined by microdilution method, the results showed four strains are sensitive to two drugs and two strains is drug resistance.In vitro exposure to stepwise increasing concentrations of enrofloxacin and norfloxacin on the four sensitive strains was used to induce drug resistance. The result showed that eight strains high degree of drug-resistant was successfully induced, MIC of the two drugs to the respective induced strain is256μg/mL.The mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes from14SS2strains of resistant bacteria were analyzed by polymerase chain reaction (PCR), the production of PCR was purified and sequenced and analyzed. The results showed that in the gyrA gene, E85→G mutation were occurred in one highly enrofloxacin-resistant strain and V68→T, S86→A, P9→W, P94→W, A98→H mutation were occurred in one highly enrofloxacin-resistant strain and one nature resistant strains. In the parC gene, E83→K mutation were occurred in three highly norfloxacin-resistant strains and two highly enrofloxacin-resistant strains, S79→I, E135→D mutation were occurred in one highly enrofloxacin-resistant strain,102H→P,118Y→H,127S→R,131L→F,139V→I mutation were occurred in one highly norfloxacin-resistan strain. With the exception of the E85→G mutation in gyrA gene and parC gene S79→I have been reported associated with drug resistance, others were not reported. And many of the highly resistant strains in this experiment did not happen to any gene mutation, indicating that the experimentally-induced resistant strains with resistance mechanism and target mutation has little relation.Resistance plasmid conjugal transfer test results showed E. coli did not get SS2of fluoroquinolone resistance plasmid. By SDS-temperature alternately eliminate plasmid of drug-resistant strains, the resistant strains, the MIC of enrofloxacin and norfloxacin have significantly decreased. By extracting the resistance plasmid of the strains before and after the eliminate plasmid test, gel electrophoresis analysis showed that resistant strains have lost the plasmid fragment.when the MIC is8μg/mL, the plasmid fragments have been all lost.In resistant strains of cross-resistance test, the results showed the exists between the cross-resistance to fluoroquinolones in S. suis resistance to different fluoroquinolones.According to the results of the initial detection of drug-resistant strains of the efflux pump like these:after1μg/mL,2μg/mL,4μg/mL,8μg/mL of different concentrations of efflux pump inhibitors cyanide chlorophenyl hydrazone (CCCP) and enrofloxacin, norfloxacin joint application, they have been reduced drug-resistant strains the MIC at8μg/mL concentration of CCCP reducing the ability of the most obvious.In order to explore whether the virulence of the SS2will change after it gets resistance. In this study, virulence factors and pathogenicity of the resistant strains were detected. The results showed that the test strains are three-sensitive parental strains and there are6strains for their respective inducible resistance. In these, the gene table type of one parental strain and its two induced strains are MRP+/EF+/SLY+/GDH-, another one parental strain and its two induced strains are MRP+/EF+/SLY-/GDH+, others are MRP+/EF+/SLY+/GDH+. Pathogenicity test results showed that the stains were highly pathogenic except a parental strain and its two induced strains which are non-pathogenic. The results indicated that the virulence factors and pathogenicity of parental strain and its induction of drug-resistant strains were the same, and did not vary because of the acquisition of resistance. It showed that the virulence did not reduce or enhance because of the generation of drug resistance. |
