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目的观察利奈唑胺是否能在体外诱导肠球菌产生高水平耐药,同时探索其耐药机制与23S rRNA的V区突变的关系,以及突变的基因拷贝数是否与耐药程度(MIC值)相关。方法 5株耐药机制未明的利奈唑胺低水平耐药粪肠球菌(MIC:4~16μg/m L)持续给予利奈唑胺进行体外诱导,观察其耐药水平变化;采用PCR技术扩增诱导菌和ATCC 29212的23S rRNA的V区片段以及编码核糖体蛋白L3、L4基因片段,分析有无V区和L3、L4突变及V区突变拷贝数。结果通过利奈唑胺持续诱导,5株耐药菌的MIC值较原菌株获得8~32倍增加,最高MIC值超过256 mg/L;其中有3株菌23S rRNA的V583区发生G2576T突变,其余2株菌未发现V区突变;所有诱导菌未发现L3、L4核糖体蛋白氨基酸改变。结论利奈唑胺在体外可诱导粪肠球菌MIC值迅速增加,其V区G2576T突变与高水平耐药密切相关,且突变拷贝数量与MIC值增高呈正相关。
Objective To observe whether linezolid can induce high level of drug-resistant enterococci in vitro and explore the relationship between its resistance mechanisms and the mutation of V region in 23S rRNA and whether the gene copy number of the mutation is related to the drug resistance (MIC) . Methods Five low-dose linezolid resistant strains of Enterococcus faecalis (MIC: 4 ~ 16μg / m L) with unknown drug resistance mechanism were treated with linezolid for induction in vitro and their drug resistance was observed. PCR amplification And the V region of 23S rRNA of ATCC 29212 and the fragments encoding the ribosomal protein L3 and L4 gene. The presence or absence of V region, L3, L4 mutation and V region mutation copy number were analyzed. Results The MIC value of 5 resistant strains was 8 ~ 32 times higher than that of the original strain by continuous induction with linezolid, the highest MIC value was over 256 mg / L. Among them, 3 strains had G2576T mutation in V583 region of 23S rRNA, No mutation in V region was found in the two strains. No amino acid changes in L3 and L4 ribosomal proteins were found in all the induced strains. Conclusion Linezolid can induce a rapid increase of MIC in E. faecalis. The G2576T mutation in V region is closely related to high level of drug resistance. The number of mutant copy is positively correlated with the increase of MIC.