Macrolide resistance by approach of gene mutation-sensitive molecular switch in Mycoplasma pneumoniae
LIU Yan1, LIU Dan2, ZHU Cui-ming3, HUANG Ze-zhi1, MENG Song-nian1, TANG Cui-lian1
1.Department of Medical Laboratory, Shaoyang Medical College, Shaoyang 422000, China; 2.Shaoyang Municipal Center for Disease Control and Prevention, Shaoyang 422000, China; 3.Institute of Pathogenic Biology, University of South China, Hengyang 421001, China
Abstract: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.
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