棒状杆菌滤膜接合法质粒接合转移实验方法的研究

目的 建立棒状杆菌的滤膜接合法质粒接合转移实验方法 .方法 采用滤膜接合法行质粒接合转移实验.第一次异种属之间的质粒接合转移实验,使用琼脂稀释法筛选出3株高耐红霉素的粪肠球菌临床分离株,红霉素最低抑菌浓度(MIC)>256 mg/L,左氧氟沙星MIC≤8 mg/L,作为供体菌;3株棒状杆菌临床分离株的红霉素MIC≤32 mg/L,左氧氟沙星MIC为128 mg/L,作为受体菌.第二次同属不同种细菌之间的质粒接合转移实验.用第一次转移成功的3株接合子干燥棒状杆菌(红霍素MIC>256 mg/L,头孢他啶MIC为16 mg/L)作为供体,分别向6株不同种的棒状杆菌(红霉索MIC≤32 mg/L,头孢他啶MIC≥128 mg/L)进行质粒接合转移.结果异种属之间的质粒接合转移实验中,3株粪肠球菌作为供体分别向3株棒状杆菌进行9次质粒接合转移,有4株接合子成功转移了耐药表型和基因型,转移频率为44%.同属不同种细菌之间的质粒接合转移实验中,3株转移成功的干燥棒状杆菌作为供体分别向6株不同种的棒状杆菌进行18次质粒接合转移,有7株接合子成功转移了耐药表型,转移频率为39%.接合子不同程度地获得了对红霉素的耐药性,棒状杆菌获得了粪肠球菌的耐药性,并凡将耐药性再传递给了同属不同种的其他棒状杆菌.结论 滤膜接合法质粒接合转移实验可以用于研究棒状杆菌的耐药传递机制.

Study on the filter mating method of plasmid conjugation experiment for Corynebacterium

Objective To set up the filter mating method of plasmid conjugation experiment for Corynebacterium.Methods The first plasmid conjugation between different genus was performed by filter method.Three strains of highly erythromycin-resistance Enterococcus faecalis isolates[minimal inhibitory concentration(MIC)of erythromycin>256 mg/L,MIC of levofloxacin≤8 mg/L] which screened by agar dilution method were used as donators and 3 strains of Corynebacteria isolates(MIC of erythromycin≤32mg/L,MIC of levofloxacin=128 mg/L)were used as acceptors.In the second conjugation between the same genus but in different species,three Corynebacterium xerosis strains(MIC of erythromycin>256 mg/L,MIC of ceftazidime=16 mg/L)which were conjugants of the first conjugation were used as donators and 6 strains of Corynebacteria isolates(MIC of erythromycin≤32 mg/L,MIC of ceftazidime 128 mg/L)were used as acceptors.Results In the first conjugation between different genus,the plasimid conjugations were done for 9 times between 3 donators and 3 acceptors,4 of which gained the phenotype and genotype of resistance and the transfer rate was 44%.In the second conjugation experiment,the plasmid conjugations were done for 18 times between 3 donators and 6 acceptors,7 of which gained the resistant phenotype and the transfer rate was 39%.Erythromycin-resistance was transferred from the donators to the conjugants in varying degrees.The resistance was transferred from Enterococcus faecalis to Corynebacteria and then to other Corynebacteria which were of the salne genus but of different species.Conclusion Filter mating conjugation method could be used to study the mechanisms of resistance transmission of Corynebacterium.