Study On Sub Inhibitory Macrolides Induced Resistance In Mycoplasma Pneumoniae Isolates

Objective: To screen sensitive strain for macrolide antibiotic from104strains ofMycoplasma pneumoniae clinical isolates preserved by institute of pathogen biologyin the university of South China，and to investigate the induction of MICconcentrations macrolide on sensitive strains induced drug resistance. And comparedmutations of Mp determining region genes between standard strains and resistantstrains induced from the clinical strains to preliminary explore the resistancemechanisms in Mp.Methods:104cases of Mycoplasma pneumoniae clinical isolates were preserved byinstitute of pathogen biology in the university of South China. The susceptibilitytesting was used to detect the MIC values of clinical isolates,and screening sensitivestrains of clinical isolates in accordance with NCCLS2006prescribes standards todetermine the sensitivity; Mycoplasma pneumoniae was induced with MICconcentrations of erythromycin, azithromycin, clarithromycin respectively for10generations. The MIC of clinical isolates after induced passaging was assayed bysensitivity test, and then compared with the corresponding MIC before to assess theinduction of macrolide induced drug resistance. The DNA template of resistantstrains that before and after induced were successfully extracted, PCR was used toamplify resistance-determining region gene, and then the PCR products weresequenced, BLAST software was used to compare sequencing results wih codingsequences and comparative analyze the gene mutation in resistance determiningregion in drug situation before and after induction.Result：(1)11cases of strains sensitive to macrolide and93cases of drug-resistant strainswere isolated from104cases of Mycoplasma pneumoniae clinical strains, so theresistant rate equaled89.4%.(2) After induced by Sub inhibitory concentration erythromycin for10generations,the MIC of8clinical sensitive strains were increased4times or more, and its resistantrate was80%. the MIC of6clinical sensitive strains were also increased4times ormore after kitasamycin induction, and its resistant rate was60%. the MIC of3clinicalsensitive strains were successfully induced to resistance for azithromycin, and its resistant rate was37.5%.(3)2of10(20%) Mycoplasma pneumoniae clinical strains exist base mutation onribosomal protein L22after resistant induction;3of10cases (30%) exsit basemutation on ribosomal protein L4; In Mp ribosomal protein L4,2cases occuredC162A mutation, A430G mutation and T279C mutation (16.66%); and1caseoccurred A209T mutation (8.33%),but base mutation on23S rRNA drug generemains not found.Conclusion(1) The macrolides resistance rate of104strains of Mycoplasma pneumoniae clinicalisolates was89.4%.(2) The resistance induced by sub inhibitory concentrations of macrolides may berelated to the ribosome L4and L22gene mutations.