肺炎支原体耐药机制的研究

发布时间：2018-02-25 09:45

本文关键词： 肺炎支原体耐药亚急性咳嗽　出处：《中国人民解放军军医进修学院》2012年博士论文　论文类型：学位论文

【摘要】：【目的】深入研究我国肺炎支原体（Mp）对大环内酯类的耐药机制和预测Mp对四环素类将可能产生的耐药机制；调研亚急性咳嗽与Mp感染的关系，以助于分析亚急性咳嗽病因学。【方法】（1）收集患者咽拭子标本，采用实时定量PCR法和液-固-液交替培养获取纯化的Mp菌株。（2）采用96孔板肉汤微量稀释法，测定大环内酯类（红霉素、阿奇霉素、麦迪霉素）、喹诺酮类（氧氟沙星、左氧氟沙星、莫西沙星）、四环素类（四环素、米诺环素）和氨基糖苷类（阿米卡星、硫酸依替米星）10种抗生素的MIC值。（3）采用巢氏PCR和多个引物一步PCR法对耐药Mp进行基因分型，并测定p1基因RepMP4和RepMP2/3序列，进行同源性分析。（4）对大环内酯类耐药株进行23SrRNA和5SrRNA基因测序，，观察外排泵抑制剂（利血平）对MIC的影响，筛查mef、msr、mph(C)等基因，进行靶位改变、外排泵和灭活酶耐药机制的研究。（5）临床筛查和体外药物诱导获取耐四环素类Mp，进行16SrRNA基因测序、筛查tet(M)基因和外排泵。（6）收集2010年7月至2011年6月军事医学科学院附属医院亚急性咳嗽患者和健康受试者咽拭子标本进行Mp检测，同时进行Mp分型、MIC测定和疗效观察，分析Mp在亚急性咳嗽中的地位和作用。【结果（】1）共获得72株Mp。（2）MIC结果显示Mp大环内酯类耐药率为41.7%（30/72）；未发现喹诺酮类耐药株；发现一株Mp对四环素类耐药；氨基糖苷类对Mp抑制作用微乎其微。（3）30株耐药株p1基因分型29株I型，1株IIa型；I型耐药菌株间存在明显同源性。（4）30株耐大环内酯Mp都存在23SrRNA2063位点突变，29株由A→G,1株为杂合子突变。一株耐药Mp中存在外排泵msrA/B基因，该基因与屎肠球菌TX2465获得的耐大环内酯类基因具有高度相似性，该基因编码的蛋白具有P-loop＿NTPase超家族相似的保守序列。未发现灭活酶mph(C)基因。（5）四环素诱导耐药株存在16SrRNA A1173T点突变，所有耐药株均未发现tet(M)基因，外排泵抑制剂（利血平）对耐药株MIC无影响。(6)亚急性咳嗽组入选83例患者，对照组80例。两组Mp检出率分别为55.4%和5%（P0.001）。患者组46株Mp基因分型I型38株（82.6%），II型6株(13.0%)，IIa型2株（4.3%）；MIC值示23株（50%）对大环内酯类耐药。Mp检测率与性别、年龄无相关，与季节有关（P=0.004），冬秋季节更易检测到Mp。观察12例耐药和18例非耐药Mp感染患者，服用阿奇霉素后咳嗽缓解天数无明显差别(P=0.671)。【结论】我国Mp对大环内酯类耐药存在靶点突变和外排泵两种机制；四环素类可体外诱导产生Mp耐药株，耐药机制可能为16SrRNA靶点突变所致； Mp感染很可能在亚急性咳嗽中发挥了重要作用。
[Abstract]:[objective] to study the drug resistance mechanism of mycoplasma pneumoniae (MP) to macrolides and to predict the possible drug resistance of MP to tetracycline in China, and to investigate the relationship between subacute cough and MP infection in order to help to analyze the etiology of subacute cough. [methods] the throat swabs of patients were collected and purified strains of MP were obtained by real-time quantitative PCR and liquid-solid-liquid alternate culture. The microdilution method of 96 hole broth was used to determine macrolides (erythromycin, azithromycin). Medelomycin, quinolones (ofloxacin, levofloxacin, moxifloxacin, tetracycline (tetracycline, minocycline) and aminoglycoside (amikacin), The MIC value of Etimixine Sulfate (10 antibiotics) was determined by nested PCR and one-step PCR with multiple primers, and the RepMP4 and RepMP2/3 sequences of p1 gene were determined. The 23s rRNA and 5s rRNA genes of macrolide resistant strains were sequenced. The effects of drain pump inhibitor (reserpine) on MIC were observed. Studies on the mechanisms of efflux pump and inactivated enzyme resistance. 5) Clinical screening and drug induction in vitro to obtain tetracycline resistant MP and sequence 16s rRNA gene. From July 2010 to June 2011, the samples of subacute cough patients and healthy subjects were collected from subacute cough patients and healthy subjects to detect MP, and the mics of MP typing and curative effect were observed. To analyze the role of MP in subacute cough. [results] A total of 72 strains of Mp.(2)MIC were obtained. The results showed that the resistance rate of MP macrolides was 41.775%, no quinolone resistant strain was found, one strain was found to be tetracycline resistant. The inhibitory effect of aminoglycosides on MP was minimal. 30 strains of drug resistant strains p1 genotyping 29 strains of type I and 1 strain of IIa type I showed obvious homology. 30 strains of macrolide resistant MP all had 23s rRNA2063 mutation and 29 strains were isolated from A. 鈫扥ne strain was heterozygous. There was an efflux pump msrA/B gene in one resistant strain, which was highly similar to the macrolide resistant gene obtained from Enterococcus faecium TX2465. The protein encoded by this gene has a conserved sequence similar to that of P-loop-NTPase superfamily. No inactivated enzyme mphCase gene, mphC5) has been found in tetracycline induced drug resistance strains. There is a 16s rRNA A1173T point mutation in all resistant strains. Extracorporeal pump inhibitor (reserpine) had no effect on drug-resistant strain MIC. In the control group, the detection rate of MP was 55.4% and 5p 0.001, respectively. There were 46 strains of MP genotyping I (38 strains) in the patient group. There was no correlation between the detection rate and sex, age, and the MIC value of 23 strains of macrolide resistance to macrolide. It was easier to detect MP in winter and autumn than that in winter and autumn. 12 cases of drug resistance and 18 cases of non-drug-resistant MP infection were observed. There was no significant difference in cough remission days after taking azithromycin (Azithromycin). [conclusion] there are two mechanisms for MP resistance to macrolides in China, such as target mutation and efflux pump, and tetracycline can induce Mp-resistant strains in vitro. The mechanism of drug resistance may be caused by 16s rRNA target mutation, and MP infection may play an important role in subacute cough.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R375

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