Application Of On/off Switches In Detecting The23S RRNA Gene Mutations And Assessing The Susceptibility Of Mycoplasma Pneumoniae To Macrolide Antibiotics

Objective: To detect the susceptibility macrolide on Mycoplasma pneumoniaeisolated from the first affiliated people’s hospital of University of South China, andthen develop an on/off molecular switch, which based on high-fidelity DNApolymerase and3′phosphorothioate-modified specific primer, to detect the23S rRNAmutants of M. pneumoniae for guiding reasonable clinical prescription during M.pneumoniae infection.Methods: M. pneumoniae pharyngeal clinical swab specimens which collectedfrom the516patients of the first affiliated hospital of University of South China, werecultured and isolated by SP4medium. Then M. pneumoniae was subcultivated in agarmedium (CM401) for single colony and was further characterized by PCRamplification. Broth microdilution method was performed to determine the minimalinhibitory concentration (MIC) of macrolides, including erythromycin, clarithromycin,azithromycin, josamycin and kitasamycin. An on/off molecular switch, which basedon high-fidelity DNA polymerase and3′phosphorothioate-modified specific primer,was used to assay the23S rRNA gene mutation sites (mainly2063,2064and2617).Gene sequencing were employed to further determine the the variation of23S rRNAgene. And the susceptibility of M. pneumoniaes to macrolide antibiotics was assessed.The correlation between23S rRNA gene mutations and macrolide resistance to M.pneumoniae was also analyzed.Results:21strains of M.pneumoniae were obtained from the516patients, thepositive rate was4.07%(51/516), and49cases were detected by PCR (9.5%,49/516).Among21M. pneumoniae strains, only2(9.5%) strains were susceptible tomacrolides, and19(90.5%) isolates were high resistant to macrolides but no moderateresistant isolates were found. Erythromycin and clarithromycin exihits highest MICs(MIC≥128mg/L) than those of azithromycin, josamycin and kitasamycin. The 15-membered macrolide azithromycin and josamycin possessed lower MIC than the14-and16-membered macrolides, and josamycin was the most susceptible drugamong the5macrolides with MIC≤4mg/L. Of the21M. pneumoniae strains,17strains were found the mutation in domain V of23S rRNA at position2063and2(9.5%) isolates were2064by on/off molecular switch. And the A2063G mutationwere found in17isolates and A2064G mutation was detected in2strains by DNAsequencing, which were in consistent with the results of molecular switch. TheA2063G/C and A2064G mutation caused M.pneumoniae strains high resistance toerythromycin.Conclusions:1. Mycoplasma pneumoniae strains isolated from the first affiliated people’shospital of University of South China were high resistant to macrolide antibiotics2．On/off molecular switches can be used to detect the23S rRNA genemutations and assess the susceptibility of M.pneumoniae to macrolide antibiotics...