Study Of Mycoplasma Pneumoniae(MP)Macrolide Resistance And Its Clinical Significance

ObjectiveMP could enter the body through the respiratory tract, causing severe respiratory disease and extrapulmonary complications. as a pathogen causing about20-30%of community-acquired pneumonia (CAP), MP has attracted the public’s attention. Recently, the increased prevalence of macrolide-resistant MP has become a significant problem because of dependence on macrolide antibiotics in clinical treatment. Some studies indicated that patients infected by macrolide-resistant MP perhaps may have heavier clinical manifestations and a longer duration of disease than that of patients infected by wild-type MP. The study was performed to investigate drug resistance of MP (including the concentrations of antibiotic resistance, macrolide resistance rates and macrolide resistance sites) and the correlation of macrolide resistance mutations with clinical manifestations.MethodsThe experimental materials were collected from Department of Respiratory Medicine and Laboratory in Children’s Hospital of Zhejiang University School of Medicine, from March to September in2013, including140respiratory specimens (including NPA, sputum samples) and112MP DNA specimens.1. Culture the MP standard strain MP FH in a variety of commercially liquid medium, solid medium under different culture conditions in order to determine the best media and culture conditions for subsequent clinical MP isolation.2. After isolated and purified, clinical MP strains were cultured by dilution susceptibility assay to determined the inhibitory concentrations of antibiotics (including macrolide antibiotics, tetracyclines, quinolones) for these strains.3. After sequencing23S rRNA domain V of the MP DNA specimens, the amplicons were used to analyse macrolide resistance rates and macrolide resistance mutation characteristics.4. Statistical analysis were exploited based on clinical data of MP-infected children, including the patient’s gender, age, diagnosis, pulmonary complications, chest X-ray images and treatment to analyse the correlation of macrolide resistance mutations with clinical manifestations.5. SPSS16.0statistical software was used for statistical analysis, data were shown as mean (x)±standard deviation (±s), two groups datas were compared using t test, count datas were compared using χ2test, P<0.05indicates statistically significant.Results1. Using the Real-Time PCR kit, there are68cases positive for MP among the140respiratory specimens, positive rate was68/140*100%=48.6%;13clinical MP strains were isolated and purified, the total isolation rate was13/140*100%=9.3%.2. The most optimal culture conditions for MP cultrue was37℃5%CO2; liquid medium Shanghai Quickda liquid medium is better than other commercial liquid medium for MP cultrue; solid medium without methylene blue and thallium acetate is more conducive to the growth of MP.3. MP which had resistance mutations showed resistant to macrolides, while was sensitive to tetracyclines and quinolones.4.105amplicons of23S rRNA domain V were obtained among112MP DNA samples, and after analysis by software,22cases existed resistance mutations in the23S rRNA domain V and macrolide resistance rate was22/105*100%=21.0%. All of the macrolide resistance mutations were A2063G mutations, other mutation sites (including2064,2067and2617sites) haven’t been found in the study.5. Among the22macrolide resistance cases, there are12cases containing part mutation in mutation site. At the same time, one mutation site G2062A that had not been reported was found. 6. Compare the clinical manifestations between macrolide resistance mutations group and wild-type MP group by statistical analysis and found that chest X-ray images of patients infected by macrolide-resistant MP is more likely to show larger shadow.Conclusions1.23S rRNA domain V mutations increased the resistant concentration of macrolides for MP strains, but changed little for that of tetracyclines and quinolones antibiotic concentration.2. Macrolide resistance mutations of MP may lead to more serious chest X-ray images of patients, with larger shadow predominant among patient infected by macrolide resistant MP, and group performance with unmutated gene patchy based.