【作者】 王长娴；

【导师】 汪宁； 王蓓；

【作者基本信息】 东南大学 ， 流行病与卫生统计学， 2004， 硕士

【摘要】 目的:(1)了解江苏省性病门诊患者淋病奈瑟菌(又称淋球菌,Neisseria gonorrhoeae)及支原体的感染状况,通过描述其分布特征,分析影响流行的主要因素。(2)对病原体流行株进行抗生素耐药性研究,为临床治疗提供科学用药的依据。(3)检测耐药基因,观察其分布状况,并分析耐药机制。研究方法:(1)应用T-M培养基分离培养淋病奈瑟菌,经Gram’s染色、氧化酶试验及API NH试剂盒进行鉴定。用法国梅里埃支原体鉴定药敏试剂盒进行解脲支原体(Ureaplasma urealyticum,Uu)与人型支原体(Mycoplasma hominis,Mh)的鉴定和药敏试验,结合流行病学调查资料,应用χ2检验、秩和检验、非条件Logistic回归进行统计分析。(2)随机抽取95株淋病奈瑟菌,采用琼脂稀释法检测头孢曲松、头孢噻肟、头孢吡肟、大观霉素、左氧氟沙星、青霉素、四环素、环丙沙星对其的最小抑菌浓度(Minimum inhibitory concentration,MIC)。(3)根据药敏结果,从95株淋病奈瑟菌中分层随机抽取48株,用PCR扩增青霉素耐药基因TEM和四环素耐药基因tetM;随机抽取20株,扩增氟喹诺酮耐药基因gyrA后测序,用DNAStar软件将标本序列与标准菌株的序列进行比对分析。分别扩增对大观霉素敏感和耐药的淋病奈瑟菌16S rRNA基因并测序分析。研究结果:(1)江苏省性病门诊就诊人群淋病奈瑟菌感染率为23.3%,解脲支原体和/或人型支原体检出率为28.9%,人型支原体主要以混合感染的形式存在。淋病奈瑟菌感染率存在地区、性别差异,徐州感染率低于南京和无锡,男性感染率高于女性。支原体检出率存在性别差异,女性检出率高于男性。(2)多因素Logistic回归分析,近期婚外性接触、性伴有性病两因素增加淋病奈瑟菌感染的机会,女性则减少感染机会。而支原体的检出率与女性及年龄小于等于25岁有关,二者增加感染支原体的风险。(3)淋病奈瑟菌对目前的一线药物(头孢类药物和大观霉素)普遍敏感,其中大观霉素的耐药率仅为1.1%。但对检测的其它药物,耐药率很高,对青霉素、四环素、环丙沙星的耐药率分别为68.4%、78.9%、100%。(4)不论单一感染还是混合感染,解脲支原体和人型支原体均对原始霉素、交沙霉素、强力霉素较为敏感,敏感率在90%以上;对环丙沙星、氧氟沙星均有不同程度耐药,且耐药率高,达40%以上。单一解脲支原体对大环内酯类药物(阿奇霉素、克拉霉素、红霉素)较为敏感,敏感率均在88%以上。但单一人型支原体对此类药物敏感率低,其耐药率均高于60%,甚至高达100%,并由此而影响支原体混合感染的耐药状况。(5)三地淋病奈瑟菌耐药谱一致。三地单一解脲支原体耐药谱一致,单一人型支原体耐药谱也基本一致(阿奇霉素除外)。(6)产青霉素酶淋病奈瑟菌(Penicillinase-producing Neisseria gonorrhoeae,PPNG)TEM基因检出率明显高于非PPNG株。tetM基因在质粒介导的抗四环素淋病奈瑟菌(Plasmid-mediated tetracycline-resistant Neisseria gonorrhoeae, TRNG)株检出率亦高于非TRNG株。而20株淋病奈瑟菌gyrA序列与标准菌株比较,存在以下四种突变形式:(1)gyrA编码的91位丝氨酸改变为苯丙氨酸和95位天冬氨酸改变为甘氨酸;(2)91位丝氨酸改变为苯丙氨酸和95位天冬氨酸改变为丙氨酸;(3)91位丝氨酸改变为苯丙氨酸和95位天冬氨酸改变为天冬酰胺;(4)91位丝氨酸改变为苯丙氨酸,92位丙氨酸改变为脯氨酸。本研究主要以91位和95位氨基酸的联合改变为主,占95%。大观霉素耐药株的16S rRNA基因第1164位发生C→T的突变。支原体的tetM基因检测显示中敏株与耐药株的检出率高于敏感株。研究结论:(1)与以往资料比较,淋病奈瑟菌与支原体感染可能有从性活跃年龄向高年<WP=5>龄组、从低文化程度向高文化程度人群扩散的趋势。(2)近期有婚外性接触史、性伴侣有性病增加淋病感染的风险, 女性由于危险行为较男性少,感染的风险也较小。而支原体检出率与女性及年龄小有关,女性及年龄小于等于25岁两因素增加支原体感染风险。(3)淋病奈瑟菌对一线药物敏感,对以往常规药物青霉素、四环素、环丙沙星耐药严重。支原体对原始霉素、交沙霉素、强力霉素敏感,对其它药物的药敏情况则因支原体种类而有所不同。(4)耐药基因在菌株中流行较广泛。淋病奈瑟菌对大观霉素耐药是由于16S rRNA基因的突变,由于细菌单一位点的突变即可引起对大观霉素的耐药,因而应重视对大观霉素的耐药监测和研究。本研究耐大观霉素株16S rRNA突变为国内首家报道, 该基因序列已成功登录美国GenBank,登录号为AY573194。更多还原

【Abstract】 Objective:(1)To study the prevalence and distribution characteristics of the Neisseria gonorrhoeae and Mycoplasma among visitors to STDs clinics in Jiangsu Province, as well as to investigate the risk factors associated with Neisseria gonorrhoeae and Mycoplasma infection. (2) To describe the resistance of the strains to the antibiotics used for treating the infections. (3)To make clear the prevalence of resistance genes among the strains. Methods: (1)Specimens from visitors were inoculated directly onto T-M selective medium for culture and incubated at 35℃ in an atmospere containing 4%~6% CO2 for 36~48hrs. Neisseria gonorrhoeae were presumptively identified on the basis of colonial and Gram’s stain morphology and positive oxidase reaction, then identity was confirmed by using API identification system.The identification and susceptibility test of mycoplasma strains was conducted by the Mycoplasma ISTⅡkit. Combining with the epidemiology data, χ2 test and Fisher’s exact test were used for comparison, and Logistic regression for finding risk factors. (2)95 strains of Neisseria gonorrhoeae were randomly selected from 125 isolates to perform the in-vitro susceptibility test. MICs of penicillin, tetracycline, ciprofloxacin, levofloxacin, ceftriaxone, cefotaxime, cefepime, spectinomycin were determined by agar dilution using GC agar base containing 1% defined supplement and serial dilutions of each agent. (3)48 strains were randomly selected from those mentioned in the susceptibility testing to amplify the TEM and tetM gene, among which, 20 strains’ gyrA gene was sequenced and compared with the sequence of ATCC19424 by DNAStar software. 16S rRNA was amplified and analyzed for the spectinomycin resistant strain and sensitive one by the same method.Results:(1)Among the visitors to STDs clinics in Jiangsu province, the prevalence of Neisseria gonorrhoeae and Ureaplasma urealyticum and/or Mycoplasma hominis were 23.3% and 28.9% respectively. The prevalence of Neisseria gonorrhoeae has significant differences between Nanjing, Xuzhou and Wuxi. The same difference was found between male and female. The prevalence of Neisseria gonorrhoeae among the visitors in Xuzhou was lower than Nanjing and Xuzhou. The infection rate among male was higher than female. The infection rate of Ureaplasma urealyticum and/or Mycoplasma hominis among female was higher than male. (2)The prevalence of Neisseria gonorrhoeae is significantly associated with gender, recent sexual contact with persons other than their own partners and the group whose partners were diagnosed with sexually transmitted diseases, while the prevalence of mycoplasma is significantly associated with female and young individuals aged less than 25y.(3)Almost all of the Neisseria gonorrhoeae strains were susceptible to the first-line antibiotics used for treating the infection. The resistance rate to ciprofloxacin, tetracycline and penicillin were 100%, 78.9% and 68.4% respectively. Most of Ureaplasma urealyticum and/or <WP=7>Mycoplasma hominis were sensitive to pristinamycine, josamycin and doxycycline. But most of them are resistant to ciprofloxacin and ofloxacin. The sensitive rate of Ureaplasma urealyticum to azithromycin, clarythromycin, erythromycin are all up to 88%, but Mycoplasma hominis are totally different.(4)TEM seems to be more frequent in PPNG than non-PPNG and tetM seems to be more frequent in TRNG than non-TRNG. 20 strains of Neisseria gonorrhoeae which are intermediate resistant and resistant to ciprofloxacin have been associated with mutations in the gyrA protein of the DNA gyrase. There are four types of alteration in the gyrA: a serine-to-phenylalanine substitution at position 91 and aspartate-to-glycine at position 95; a serine-to-phenylalanine substitution at position 91 and aspartate-to-alanine at position 95; a serine-to-phenylalanine substitution at position 91 and aspartate-to-asparagine substitution at position95; a serine-to-phenylalanine substitution at position 91 and alanine-to-proline at position 92. The strain resistant to spectinomycin is更多还原