解脲脲原体与淋球菌耐药表型及基因型的流行病学研究

发布时间：2018-04-16 23:36

本文选题：解脲脲原体 + 淋球菌　；参考：《东南大学》2006年硕士论文

【摘要】： 目的:(1)调查江苏省局部地区解脲脲原体的耐药状况并提出用药建议;(2)研究江苏省局部地区解脲脲原体的耐药机制(包括对四环素类、氟喹诺酮类和大环内酯类药物);(3)根据解脲脲原体两个不同基因型的基因组差别建立和评价新的基因分型方法,并进行初步的临床应用;(4)研究江苏省淋球菌对氟喹诺酮类药物的耐药机制。研究方法:(1)用法国梅里埃支原体鉴定试剂盒进行解脲脲原体(Uu)与人型支原体(Mh)的初步分离和鉴定,并对解脲脲原体培养阳性物进一步经过滤培养和特异性PCR试验进行确认。(2)应用微量肉汤稀释法检测八种抗生素(四环素、米诺环素、红霉素、克拉霉素、阿齐霉素、环丙沙星、氧氟沙星和左旋氧氟沙星)对确认的84株解脲脲原体的最小抑菌浓度(Minimum inhibitory concentration,MIC)。(3)按药敏结果分层,从84株解脲脲原体菌株中随机抽取34株,用PCR扩增四环素耐药基因tetM;随机抽取28株,采用PCR扩增氟喹诺酮耐药基因gyrA的氟喹诺酮耐药决定区(QRDR),并对扩增产物进行测序分析,采用Blast比对法比较临床菌株序列与标准菌株序列的差异。(4)基于解脲脲原体14个血清型的23S rRNA基因序列的差异,采用primer premier5.0软件设计PCR引物和选取限制性内切酶,自行建立解脲脲原体的基因分型方法,通过与标准菌株和“金标准”鉴定的临床菌株比较,对其进行评价并初步应用于临床菌株进行基因分型和分析。(5)根据淋球菌药敏结果,从已经鉴定为淋球菌的95株菌株中,分层随机抽取54株,采用PCR扩增氟喹诺酮耐药基因gyrA和parC的QRDR,并进行DNA测序,分析其突变位点与药敏结果MIC的相关关系。(6)结合实验结果和流行病学调查资料,采用卡方检验、方差分析、秩和检验和精确概率法对数据进行统计分析。研究结果:(1)解脲脲原体对米诺环素、四环素、克拉霉素、阿齐霉素、红霉素、环丙沙星、氧氟沙星和左旋氧氟沙星的MIC50(50%的菌株被抑制的浓度)分别为0.0625、0.125、0.25、1、2、8、4和2mg/L;MIC90(90%的菌株被抑制的浓度)分别为0.125、1、1、4、8、64、16和8mg/L。(2)大环内酯类抗生素(克拉霉素、阿齐霉素和红霉素)的耐药水平(包括MIC几何均数和耐药率)在有Mh合并感染组与无Mh合并感染组间有统计学差异(P0.05),伴随Mh感染的解脲脲原体对大环内酯类抗生素的MIC水平和耐药率都较高。(3)在34株非四环素耐药的解脲脲原体菌株中,有7株检测到tetM基因。tetM基因阳性组的四环素和米诺环素的MIC水平都比tetM阴性组高,且有统计学意义(四环素:t=-4.34,P=0.0001;米诺环素:t=-5.90,P0.0001)。在28株解脲脲原体菌株中,经测序分析发现gyrA基因出现三种突变形式:①302位碱基C→T的突变,导致101位(相当于大肠杆菌84位)丙氨酸改变为缬氨酸;②336位碱基C→A的突变,导致112位(相当于大肠杆菌95位)天冬氨酸改变为谷氨酸;③267位碱基T的缺失。发生gyrA基因突变的主要是耐药菌株和中度敏感菌株,在敏感菌株没有发现gyrA基因突变;所有有基因改变的菌株其氟喹诺酮类药物的MIC水平明显高于没有突变的菌株组。(4)在标准菌株中,PCR-RFLP(PCR为基础的限制性酶切多态性分析)方法分型能力为100%,同时分型结果有较好的特异性和可重复性,电泳图谱清晰易于分辨和解释。在通过“金标准”鉴定的临床菌株中,该分型方法的灵敏度为97%,特异度为89%,重复试验一致率为98%。PCR阳性扩增模板两次独立分型实验的一致率为90.62%;PCR-RFLP分型结果与测序分型结果的一致率为100%。(5)有生殖道感染病史和合并Mh感染的解脲脲原体基因型主要表现为生物1型,而合并霉菌感染的解脲脲原体主要为混合型。不同型别的解脲脲原体对四环素类、大环内酯类和氟喹诺酮类药物的敏感性存在统计学差异,其MIC的平均水平均表现为T960型高于生物1型。解脲脲原体基因型与氟喹诺酮类药物耐药相关的gyrA基因的氨基酸改变有较强的相关性,出现氨基酸Asp95Glu改变的菌株均属于T960型,而无此改变的菌株均不属于T960型。(6)分离的95株淋球菌对环丙沙星100%的耐药;通过测序分析,在54株淋球菌中检测到gyrA和parC的18种突变形式。环丙沙星的MIC水平在不同parC基因突变组之间差异有统计学意义(P=0.006),parC基因突变位点越少其MIC相对较低,突变位点越多其MIC相对较高。研究结论:(1)在江苏局部地区第三代以前的氟喹诺酮类药物和红霉素MIC水平较高,耐药严重,此类药物不宜作为治疗解脲脲原体感染的一线药物,而四环素类药物可以重新作为治疗解脲脲原体感染的一
[Abstract]:Objective: (1) the status of drug resistance investigation of Ureaplasma urealyticum in some areas of Jiangsu province and put forward recommendations; (2) the resistant mechanisms in some areas of Jiangsu province of Ureaplasma urealyticum (including tetracyclines, fluoroquinolones and macrolides); (3) according to the genomic difference of Ureaplasma urealyticum in two different genes the establishment and evaluation of a new genotyping method and preliminary clinical application; (4) the mechanism of fluoroquinolone resistance of Neisseria gonorrhoeae in Jiangsu province.
Research methods: (1) Ureaplasma urealyticum Mycoplasma in France bioMerieux identification Kit (Uu) and human Mycoplasma (Mh) preliminary isolation and identification of Ureaplasma urealyticum, and positive culture further filtered culture and specificity of PCR test were confirmed. (2) using broth dilution method to detect eight kinds of antibiotics (tetracycline, minocycline, erythromycin, clarithromycin, azithromycin, ciprofloxacin, ofloxacin and levofloxacin) the minimum inhibitory concentration of 84 strains of Ureaplasma urealyticum (Minimum inhibitory confirmed by concentration, MIC). (3) stratified by drug sensitivity test, were randomly selected from 84 strains of Ureaplasma urealyticum strains in 34 strains. Amplification of tetracycline resistant gene tetM with PCR; randomly selected 28 strains of fluoroquinolone resistance by PCR amplification of fluoroquinolone resistance gene gyrA (QRDR), and determine the area of amplified products were sequenced by Blast analysis, comparison method comparison The difference of clinical strains and standard strains sequence sequence. (4) the difference of 23S rRNA gene sequence of Ureaplasma urealyticum serotype 14 based on primer, using premier5.0 software to design PCR primers and selected restriction enzymes, to establish Ureaplasma urealyticum genotyping method through the comparison of clinical strains and standard strains and gold the standard "identification, evaluate and preliminary clinical application of strain type and gene analysis. (5) according to the results of drug sensitivity of Neisseria gonorrhoeae, from have already identified 95 strains of Neisseria gonorrhoeae, were randomly selected from 54 strains of fluoroquinolone resistance and gyrA gene amplification by parC with PCR and QRDR. The sequence of DNA, to explore the relationship between the mutation and susceptibility of MIC. (6) according to the experimental results and epidemiological data, using the chi square test, analysis of variance, statistical data and rank test and exact probability method Analysis.
Results: (1) of Ureaplasma urealyticum to tetracycline, minocycline, clarithromycin, azithromycin, erythromycin, ciprofloxacin, ofloxacin and levofloxacin (MIC50 concentration of 50% strains was inhibited by 0.0625,0.125,0.25,1,2,8,4 and 2mg/L respectively); MIC90 (concentration of 90% strains inhibited) were 0.125,1,1,4,8,64,16 and 8mg/L. (2) macrolide antibiotics (clarithromycin, azithromycin and erythromycin resistance (MIC) level including geometric mean and resistance rate) in Mh infection group and Mh infection group had statistically significant difference (P0.05), with Mh of Ureaplasma urealyticum infection of macrolide antibiotics the level of MIC and the resistance rate is higher. (3) in Ureaplasma urealyticum strains 34 strains of non tetracycline resistance in 7 strains were detected tetM gene.TetM gene positive group of tetracycline and minocycline MIC levels than tetM negative group, and There was statistical significance (t=-4.34, P=0.0001; tetracycline, minocycline: t=-5.90, P0.0001). In the 28 strains of Ureaplasma urealyticum strains, by sequencing the gyrA gene mutation of three mutated forms: 302 base C to T, resulting in 101 (equivalent to Escherichia coli 84) alanine to valine change; the 336 base mutation of C to A, resulting in 112 (equivalent to Escherichia coli 95) aspartate to glutamic acid; lack of the 267 base T. GyrA gene mutation is the main drug resistant strains and moderately sensitive strains in the sensitive strains, no mutation of the gyrA gene; all the gene change the strain of the fluoroquinolone MIC level is significantly higher than that without strain group mutation. (4) in the standard strains, PCR-RFLP (restriction enzyme polymorphism analysis PCR based typing method cut) was 100%, the specificity is good at the same type of results And repeated, clear and easy to distinguish and explain the electrophoresis. In clinical strains through the "gold standard" identification, sensitivity of the typing method was 97%, the specificity was 89%, the coincidence rate is 98%.PCR positive repeat test two independent template classification experiments the concordance rate was 90.62%; the consistent rate of PCR-RFLP the results and sequencing typing results for 100%. (5) with reproductive tract infection of Ureaplasma urealyticum genotypes with Mh infection history and mainly for biological type 1, and the combination of Ureaplasma urealyticum fungal infection is mainly mixed type. Different types of Ureaplasma urealyticum to tetracyclines, there was significant difference in sensitivity of ring macrolides and fluoroquinolones, the average level of MIC were higher than the T960 type biological type 1. Amino acid of gyrA gene of Ureaplasma urealyticum genotypes associated with fluoroquinolone resistance is stronger than the phase change The correlation of the amino acid change of Asp95Glu strains belong to the T960 type, but not change the strain does not belong to T960 type. (6) of 95 strains of Neisseria gonorrhoeae isolates resistant to ciprofloxacin 100%; through sequencing, 18 mutations gyrA and parC was detected in 54 strains of Neisseria gonorrhoeae to ciprofloxacin. The level of MIC in different parC mutation group have statistically significant difference (P=0.006), parC gene mutation in the less the MIC is relatively low, the more the MIC mutation is relatively high.
Research conclusions: (1) in the third Jiangsu local area before the generation of fluoroquinolones and erythromycin MIC high level of resistance is serious, that these drugs should not be used as first-line treatment of Ureaplasma urealyticum infection, and tetracycline treatment can be used as a primary infection of Ureaplasma urealyticum

【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R181.3

【参考文献】