【作者】 于军；

【导师】 吴移谋；

【作者基本信息】 南华大学 ， 病原生物学， 2006， 硕士

【摘要】 目的 通过检测人型支原体（Mycoplasma hominis，Mh）临床株对5种大环内酯类抗生素的敏感性，研究耐交沙霉素Mh临床株23S rRNA Ⅱ区和Ⅴ区核苷酸突变与对大环内酯类抗生素耐药性的关系，为临床治疗提供实验依据，以了解Mh对大环内酯类抗生素的耐药机制。 方法 应用微量肉汤稀释法检测106株Mh对5种大环内酯类抗生素的敏感性，以MIC值大于或等于标准敏感株的4倍为参考标准，从中筛选临床耐药株。按照文献设计并合成耐交沙霉素Mh临床株23S rRNA Ⅱ区和Ⅴ区DNA序列的引物，利用多聚酶链反应（PCR）扩增其DNA片段并测序。通过Blast软件进行分析，将其与Mh标准敏感株PG21核苷酸序列进行比较，分析核苷酸突变及其与大环内酯类抗生素耐药性的相关性。 结果 在106株Mh临床株中，对5种大环内酯类抗生素的敏感性以交沙霉素抗Mh活性最强，MIC₅₀为0.125mg／L，MIC₉₀均为0.5mg／L，其次为罗红霉素、克拉霉素、阿奇霉素MIC₅₀分别为4mg／L、32mg／L、64mg／L，MIC₉₀分别为32mg／L、128mg／L、512mg／L，红霉素的抗Mh活性最差，其MIC₅₀和MIC₉₀分别达到64mg／L和512mg／L。抑制100％的Mh临床株交沙霉素只需4mg／L，而罗红霉素需64mg／L、克拉霉素、阿奇霉素、红霉素需512mg／L。 利用引物经PCR扩增耐交沙霉素Mh临床株的23S rRNA Ⅱ区和Ⅴ区，测序后Blast软件分析得出：23S rRNAV区2059位发生A→G突变，2610发生C→T突变，2670和2675位发生G→T突变，2687位发生G→A突变。耐交沙霉素Mh临床株更多还原

【Abstract】 Objective: To provide experimental data for clinical chemotherapy and understand the resistance mechanism of Mycoplasma hominis (Mh) to macrolides, the susceptibility of Mh clinical isolates to 5 macrolides was detected, and the correlation of nucleotide mutations in domain II and domain V of 23S rRNA and the resistance to josamycin in Mh clinical isolates was studied.Method: Susceptibility tests of 106 Mh clinical isolates to 5 macrolides were performed by using broth microdilution method ,according to the criteria of MICs being 4-fold or higher than the susceptible reference strain’s counterparts. The specific primers of domain II and domain V of 23S rRNA of Mh clinical isolates resistant to josamycin were synthesized and DNA fragments were amplified by polymerase chain reaction(PCR) and amplicons were sequenced. The nucleotide sequences of Mh clinical isolates resistant to josamycin were compared with the reference strain’s counterparts with Blast software, and the correlation of nucleotide mutation with the resistance to macrolides was analyzed.Results: In 106 Mh clinical isolates to 5 macrolides , josamycin was the most active agent against Mh, the MIC50 being 0.125 mg/L, MIC90 being 0.5 mg/L, roxithromycin, clarithromycin and azithromycin were less active against Mh, the MIC50 being 4 mg/L,32 mg/L and 64 mg/L, respectively; MIC90 being 32 mg/L,128 mg/L and 512 mg/L, respectively. Eryhthromycin had the least activity against Mh,with MIC50 and MIC90 being 64mg/L and 512 mg/L. Based on the accumulated inhibit bacteria rate at each concerntration, 4mg/L was required to inhibit 100% of Mh for josamycin ,whereas inhibit 100% of Mh required 64 mg/L for roxithromycin and required 512 mg/L for clarithromycin, azithromycin and erythromycin.Amplified DNA fragments of Mh clinical isolates resistant to josamycin in domain II and domain V of 23S rRNA by PCR. After sequencing, the DNA sequence更多还原