【作者】 毛昱；

【导师】 孟冬娅；

【作者基本信息】 大连医科大学 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的:支原体(Mycoplasma)是引起泌尿生殖道感染较为常见的病原体,生殖道支原体感染往往引起非淋病性尿道炎(NGU)、宫颈炎、盆腔炎、自发性流产、不孕等严重后果。部分病人感染以后由于治疗不及时或治疗方法不得当,经常会导致病情反复发作和支原体对药物产生耐药性,给临床治疗带来困难。经过临床的检测及药敏试验可以发现,虽然支原体感染以解脲脲原体为主,但感染人型支原体(Mh)的病人多表现为病史较长,且Mh药敏结果为多重耐药。支原体由于缺乏细胞壁,故对于干扰细胞壁形成的l3一内酰胺类抗生素,如青霉素、头孢菌素等具有天然的耐药性;又因在Mh基因组中携带有耐四环素的tetM基因,从而使四环素的应用受到局限,现在临床上用于治疗人型支原体感染的主要为大环内酯类药物和喹诺酮类。本研究对临床分离出的支原体感染标本进行了药敏试验,并对其耐药情况进行了分析,同时对人型支原体对大环内酯类药物的耐药机制进行了探讨,力求找出其耐药的根本因素,从而能够为临床治疗及耐药机理的研究提供理论依据。方法:从2003年1月至2006年12月来我院皮肤科和妇科门诊就诊患者共4846例为统计对象,其中男1726例,女3120例,年龄2～69岁。通过珠海丽珠公司提供的培养药敏试验板对其进行药敏试验,统计其耐药情况。经过液体培养基分离培养,固体培养基鉴定并纯化的方法进行筛选,从上述标本中筛选出20株人型支原体,排除了了其他细菌及支原体感染的可能。应用PCR对可能导致耐大环内酯类药物Erm A、Erm B、Erm C以及Mef A基因的检测,并对人型支原体与大环内酯类药物的结合位点所在区域23 s rRNA的V区进行了序列的扩增,扩增产物通过琼脂糖凝胶电泳进行鉴定。结果:临床上以解脲脲原体感染居多,约占NGU的40%,混合感染次之,单纯人型支原体感染较少;Mh对14、15环大环内酯类药物耐药情况比较严重,对16环交沙霉素敏感性较高。20例菌株中未发现ErmA、B、C的阳性条带;Mef A的检测也未发现阳性条带。对23S rRNA的V区扩增的结果与基因Bank中的人型支原体序列比较显示,对交沙霉素敏感株未发生位点突变,同时还和肺炎支原体序列进行了比对,结果发现在2057位可发现G-A的改变。经十六环大环内酯类药物诱导产生耐药的两株人型支原体最小抑菌浓度分别为64mg/l和128mg/l,两个诱导株中的2059位存在着A-G的突变。结论:对于混合感染病例的病例,应排除用14、15环大环内酯药物进行治疗,以防止消除Uu感染后仍存在Mh感染.人型支原体对大环内酯类药物的耐药主要可以归结为药物结合位点的改变,而并非甲基化酶以及外排泵作用,对16元环大环内酯类药物获得性耐药的菌株,其机制可能与2059位的A-G突变导致支原体与药物结合能力下降有关。更多还原

【Abstract】 IntroductionHuman mycoplasmas are common pathogens of urogenital and respirat- ory tract infections. They can cause non-gonococcal urethritis、cervicitis、pelvic inflammatory disease、spontaneous abortion、infertility and other diseases. Due to unsuitable treatment after infection,more and more drug-resistance had been reported recent years .we also can find that the resistance of mycoplasmas to microlide are increasing year by year by the detection of susceptibility test. It was even worse in the mixed infection by Mycoplasma hominis(Mh) and ureaplasma urealyticum(Uu) and Mh alone.Mh is one of the main pathogens of urogenital tract infections, some antibiotics such as penicillin、cephalosporin are inefficacious to it because it lacks of cell wall;and also there is Tet M gene in its genome, tetracycline are inefficacious too. In clinical, doctor usually use macrolides and quinolones.In this research, we had analysised the drug- resistance conditions of the clinical samples by susceptibility test , meanwhile, we had explored the mechanism of drug-resistance of Mh to the. Macrolides.MethodsUrogenital specimens from 4846 (male:1726,female:3120, age:2-69) outpatients were assayed by the susceptibility test according to the instructions of kits which were provided by Lizhu company.To choose 20 Mh specimens from above metioned samples for the research of drug-resistance. All of them are cultured in liquid medium and purified by the solid medium to exclude other microorganisms.To assay the Erm A、Erm B、Erm C and Mef A by polymerase chainreaction and the domain V of 23s rRNA where the binding site of Mh to macrolides are also amplified. The product are detected by agarose gel electrophoresisComparing the sequence of the products of 23s rRNA with correspon- ding sequence in genebank ,and analysis the mutation .ResultsInfections by Uu are common in clinical, it occupied 40% of NGU, and infection by Mh alone are rare appearance, all of them were mearly resistance to the 14、15 ring macrolides but sensitive to 16 ring macrolide, Josamycin. In mixed infection cases, it should be treated without 14、15 ring microlide to prevent the Mh infection coming worse.In this research, we didn’t find the Erm A、Erm B、Erm C and Mef A gene after PCR detection.After comparing to sequence of Mycoplasma pneumonia Genebank ,we can find a G2057A mutation.Refer to the induced resistant strain, the MIC is 64mg/L and 128mg/L respectively.In this research, we can find a A2059G mutation in common. ConclusionThe resistance of Mh to macrolides are not caused by the methylase (ErmA、Erm B、Erm C) and Mef A but caused by the mutant of binding site to the drug. And to acquired resistance to 16 ring macrolide, the mechanism may be the A2059G mutant in 23s rRNA domain V.更多还原