突变敏感性分子开关检测肺炎支原体对大环内酯类抗生素的耐药性研究

目的 应用突变敏感性分子开关检测肺炎支原体对大环内酯类抗生素的耐药性。方法 采用微量稀释法检测5种常用大环内酯类抗生素对40株Mp临床分离株的药物敏感性;建立高保真聚合酶和3'硫化修饰引物的分子开关,用分子开关进行Mp临床分离株的PCR扩增,检测其是否存在Mp 23S rRNA 2063、2064和2617 3个热点突变,并通过基因测序进一步确定是否存在点突变,分析点突变与大环内酯类抗生素敏感性之间的关系。结果 5种大环内酯类抗生素中,Mp对14元环的红霉素和克拉霉素耐药程度最高,其MIC≥128 mg/L;对15元环的大环内酯类抗生素阿奇霉素和交沙霉素相对敏感,其中交沙霉素抗Mp活性最高,其MIC≤4 mg/L。用高保真聚合酶和3'硫化修饰引物的分子开关行PCR扩增,检测出35株发生了2063位点基因突变,3株发生2064位点基因突变,未检测出2617位点突变。用基因测序检测到35株Mp发生A2063G的突变,3株发生A2064G的突变,未检测到2617位点突变,与分子开关的检测结果一致,并且2063、2064位点突变Mp株均对大环内酯类药物高度耐药。结论 分子开关可识别23S rRNA基因突变,可用于分析Mp对大环内酯类抗生素的敏感性。

Macrolide resistance by approach of gene mutation-sensitive molecular switch in Mycoplasma pneumoniae

We detected macrolide resistance in Mycoplasma pneumoniae (M. pneumoniae) with approach of gene mutation-sensitive molecular switch. Broth microdilution method was performed to determine the minimal inhibitory concentration (MIC) of macrolides for 40 strains of M. pneumoniae. An on/off molecular switch, which based on high-fidelity DNA polymerase and 3' phosphorothioate-modified specific primer, was developed to assay the 23S rRNA gene mutation sites (maily 2063, 2064 and 2 617) of M. pneumoniae isolates. Gene sequencing were employed to further determine the variation of 23S rRNA gene, and the susceptibility of M. pneumoniaes to macrolide antibiotics was assayed. Results showed that among 40 strains of M. pneumoniae, 38 (95.00%) isolates were high resistant to macrolides. Erythromycin and clarithromycin had higher MICs (MIC≥128 mg/L) than those of azithromycin, josamycin and kitasamycin. The 15-membered macrolide azithromycin and josamycin possessed lower MIC than the 14- and 16-membered macrolides, and josamycin was the most susceptible drug among the 5 macrolides with MIC ≤4 mg/L. In 40 strains of M. pneumoniae, 35 isolates were detected the mutation in domain V of 23S rRNA genes at position 2 063 and 3 isolates were detected the mutation at position 2 064 by on/off molecular switch. And the A2063G transition in domain V of 23S rRNA genes was found in 35 isolates and A2064G mutation was detected in 3 strains by DNA sequencing, which were consistent with the results of molecular switch. The A2063G/C and A2064G mutation caused erythromycin resistance in M. pneumoniae strains. It’s suggested that on/off molecular switches could be used to detect the 23S rRNA gene mutations and assess the susceptibility of M. pneumoniae to macrolide antibiotics.