The Initial Study On Antimicrobial-resistance Gene Of Mycoplasma Pneumoniae And Its Clinical Significance

Background:Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP) is common community-acquired respiratory tract infections in children and adolescents of common pathogens, mainly spread through respiratory tract. MP infection can cause upper respiratory tract infections, bronchitis, pneumonia, can also cause serious extrapulmonary complications, such as immune hemolytic anemia, meningitis, myocarditis, nephritis. MP pneumonia incidence on the rise in recent years, and refractory or severe MP pneumonia cases increasing, which did great harm to immune compromised population such as children and the elderly poor immunity.MP are cell wall-free organisms. Their sizes are between viruses and bacteria. They are the smallest prokaryotic microbes that are capable of growing in lifeless media. MP effect on the bacterial protein synthesis of macrolide antibiotic classes, quinolone, aminoglycosides, tetracycline class sensitive. As most of the drugs have adverse reactions on the children's normal growth and development, currently macrolide antibiotics is a drug of first choice for treatment of Mycoplasma pneumonia in children. With macrolides widely used in clinical, drug resistance of MP is also emerging.MP on drug resistance macrolide erythromycin is the main molecular basis of target 23S rRNA gene 2063 and 2064 point mutations. We application PCR spread increased pneumonia support original body 23S rRNA v regional easy occurs mutation of gene fragment. The DNA sequences are compaired to the sequence of MP to find molecular mechanism of drug resistance. And on MP pneumonia in children with of clinical information for statistics analysis, to understanding MP of resistance drug and MP of gene mutation and MP pneumonia of clinical correlation.Objectives:To understand the MP status and drug resistance genes and MP pneumonia of clinical relevance.Methods:235 nasopharyngeal aspirate (NPA) were collected from children with MP pneumonia hospitalized in Children's Hospital of Zhejiang University school of Medicine during pneumonia the period from April in 2009 to April in 2010. The reseach is to detect and sequence MP 23 S rRNA gene and to analysis clinical data.1. Collected the NPA clinical specimens from children with MPP; and isolated the DNA of MP for fluorescence quantitative PCR. We selected the specimens whose concentration was 1.0*10∧4or above (includingl.0*10∧4), and then drawn the supernatant after 4000rpm/15min centrifugation to 1.5ml eppendorf tube, frozen the supernatant in-80℃refrigerator.2. Application of PCR for detecting MP 23S rRNA gene:from the United States national biological information (NCBI) MP retrieved 23S rRNA gene sequences, based on our and foreign research reports mutation Hotspot region on both sides of 23S rRNA gene 2063 and 2064 point to design PCR primers, to detection of the amplification products, purpose to detect the presence of fragments.3. Apply DNA sequencing technology detection 23S rRNA gene:detection of positive specimens of the purification of alcohol after end of the dideoxy termination method for automated DNA sequencing, sequences and NCBI logged MP standards measured strain (M129) 23S rRNA genes than on each test set up positive and negative control.4. Collecting and comparing MP the 23S rRNA gene mutation and mutation does not occur groups MP of gender, age, clinical manifestations, complications of pneumonia, laboratory results, characteristics and treatment of chest x ray, and so on.Result:1. NPA-DNA fluorescence probe by PCR method for determination of concentration of more than 1.0*10∧4 (including 1.0*10∧4) 235 specimens.2. MP samples using PCR for direct detection of 235 cases of eryt hromycin target 23S rRNA genes are positive. The 23S rRNA gene was amplified and the gene sequence was compaired with MP reference strain in Genbank.26 cases gene sequences were indentical to the reference strain.206 cases gene had point mutations, the percentage of point mutations was 87.7%.3.206 cases gene had gene point mutations in 23S rRNA, Among them,199 had A to G mutation at position 2063,6 had A to T mutation at position 2063,1 had A to G mutation at position 2064.4. Clinical data analysis found, MP pneumonia of 23S rRNA gene point mutation and mutation MP pneumonia does not occur, both outside the chest imaging, pulmonary complications, the average time of average hospital stay, fever, and the use of large ring macrolide antibiotics average cooling time after treatment there are significant differences.Conclusion:1. The percentage of point mutations in 23S rRNA was 87.7% during pneumonia the period from April in 2009 to April in 2010.The percentage of point mutations at position 2063 in 23S rRNA point mutations was 99.5%. It can be seen 2,063 dominated locus mutation.2. MP resistance genes, clinical manifestations were more serious, long course, easier to appear outside the lung damage and refractory.