【作者】 徐金水；

【导师】 王蓓；

【作者基本信息】 东南大学 ， 流行病与卫生统计学， 2006， 硕士

【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(P<0.05),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,P<0.0001)。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更多还原

【Abstract】 Objective: (1) To determine the status of eight antimicrobial agents resistance among U.urealyticum strains in Jiangsu Province. (2) To study the mechanisms of U.urealyticum resistance to common used antimicrobial agents including tetracycline, fluoroquinolones and erythromycin. (3)To design and evaluate the PCR-RFLP genotyping method according to the difference of 23S rRNA between Biovar 1 and T960 types, and to further investigate the association of these biovars with gynecological coinfections and susceptibilities of U.urealyticum to antibiotics. (4)To study correlation of in vitro susceptibilities to fluoroquinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC. Methods:(1)The preliminary isolation and identification of mycoplasma strains including U.urealyticum and Mycoplasma hominis were conducted by Mycoplasma ID kit. The positive specimens of U.urealyticum were further identified by culture of filtered positive medium and specific PCR assay. (2)In-vitro susceptibilities of U.urealyticum (MICs) to eight antimicrobial agents (tetracycline, minocycline, erythromycin, clarythromycin, azithromycin, ciprofloxacin,ofloxaxin and levofloxacin) were determined by a broth microdilution method. (3)According to the susceptibilities of U.urealyticum,34 strains of U.urealyticum were randomly selected from 84 isolates to amplify the tetM gene, specific fragments of gyrA genes of randomly selected 28 strains were amplified and sequenced. Blastn program was used to analyze and compare the sequence of clinical isolates with the sequence of standard strain T3. (4) Primer premier5.0 software was used to design PCR assay and select appropriate restricted endonuclease according to the difference of 23S rRNA gene between Biovar 1 and T960 types, new genotyping method was designed and evaluated by comparing its genotyping results with standard strains and clinical strains identified by“golden standard”. (5) 54 strains of N.gonorrhoeae were randomly selected from 95 clinical isolates, chromosomal DNA of which was used as a template in PCR to amplify the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes. And PCR-amplified DNA was further sequenced. (6) In combination with epidemiological data,experimental results were analyzed by usingχ2 test, fisher’s exact test and ANOVA analysis method.Results: (1)For U.urealyticum the MIC50 of minocycline, tetracycline, clarythromycin, azithromycin, erythromycin, ciprofloxacin, ofloxacin and levofloxacin were <0.0625, <0.125, 0.25, 1, 2, 8, 4 and 2mg/L respectively; the MIC90 of minocycline, tetracycline, clarythromycin, azithromycin, erythromycin, ciprofloxacin, ofloxacin and levofloxacin were 0.125, 1, 1, 4, 8, 64, 16 and 8mg/L respectively. (2) Significant difference of susceptibilities of U.urealyticum to macrolide were found between Mh coinfection group and non-Mh coinfection group, U.urealyticum from Mh coinfection group showed higher resistance to macrolide. (3) TetM gene was detected among 7 of 34 U.urealyticum strains, strains with tetM gene showed more resistance to tetracycline and minocycline in comparison with strains with non-tetM gene. Three alterations of GyrA in 28 U.urealyticum strains were found:①A alanine to valine substitution at position 101(corresponding to position 84 in E.coli);②A aspartic acid to glutamine substitution at position 112(corresponding to position 95 in E.coli);③An absence of T base at position 267 in gyrA gene. U.urealyticum with GyrA alterations was more resistant to fluoroquinolones and GyrA alterations were not found among all strains susceptible to fluoroquinolones. (4) PCR-RFLP typing method had excellent typeability(100%), specificity and repeatability among standard strains, the map of agarose gel electrophoresis was clear and easy to differentiate and interpret. Among clinical isolates identified by“golden standard”, the sensitivity and specificity of PCR assay were 97% and 89% respectively,更多还原