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目的体外诱导替加环素耐药金黄色葡萄球菌的核糖体16Sr RNA基因位点变异研究。方法收集4株替加环素均敏感的金黄色葡萄球菌,分别编号为S2、S3、MS2和N315,通过体外不同浓度梯度多步诱导替加环素耐药;挑取单克隆,经BD公司viet自动药敏分析仪分析常规药敏特点;用E-test条测定母株和诱导菌株替加环素MIC值,并用琼脂平板稀释法测定诱导系列Omacycline和Eravacycline的MIC值;提取耐药菌株基因组DNA,PCR扩增金黄色葡萄球菌5个拷贝的16Sr RNA基因,扩增产物经测序后与野生株比较获得突变位点。结果经体外多步法诱导替加环素耐药的不同MIC值的金黄色葡萄球菌共8株。PCR测序分析4株母株均无变异位点,16Sr RNA的突变位点主要是C1036T、G848C,此外还有T1043C、T1125C、T1038G、A1053G、T1283C、C1042T、C1047T、G1035A、C817G、G1107C、G697T、C819G。结论体外多步法可诱导金黄色葡萄球菌替加环素耐药,耐药机制可能与16Sr RNA位点发生C1036T和G848C突变相关,但仍需进一步研究证实突变位点与耐药的关系。
Objective To study ribosomal 16S rRNA gene locus variation induced by tigecycline - resistant Staphylococcus aureus in vitro. Methods Four strains of Staphylococcus aureus sensitive to tigecycline were collected and identified as S2, S3, MS2 and N315 respectively. Tigecycline was induced by multi-step in vitro with different concentration gradient. viet automatic susceptibility analyzer analysis of conventional drug-susceptibility characteristics; with the E-test strip determination of genetically modified strains of tigecycline and the MIC values and agar plate dilution method for the determination of the series of Omacycline and Eravacycline the MIC value; DNA, PCR amplification of 5 copies of Staphylococcus aureus 16Sr RNA gene amplification products were sequenced and compared with the wild-type mutants obtained site. Results A total of 8 strains of Staphylococcus aureus with different MICs were obtained by multi-step in vitro induction of tigecycline resistance. There were no mutation sites in the 4 strains of mother plants by PCR analysis. The mutation sites of 16S rRNA were mainly C1036T and G848C, in addition to T1043C, T1125C, T1038G, A1053G, T1283C, C1042T, C1047T, G1035A, C817G, G1107C, G697T, C819G. Conclusion The multi-step in vitro method can induce the resistance of TiCl4 to Tigecycline in Staphylococcus aureus. The mechanism of drug resistance may be related to the mutation of C1036T and G848C at 16Sr RNA site. However, further study is needed to confirm the relationship between the site of mutation and drug resistance.