淋病奈瑟菌外膜蛋白PorB基因的克隆及原核表达

目的:构建淋病奈瑟菌外膜蛋白PorB的融合表达载体,并在原核系统中表达,获得基因重组蛋白,为进一步研究淋病奈瑟菌外膜蛋白的致病作用和免疫保护性提供基础。方法:根据PorB已知序列设计一对引物,用PCR方法从淋病奈瑟菌标准菌株NG 29403基因组DNA中扩增出PorB基因,与原核表达质粒pGEX-4T-1构建重组子pGEX-4T-PorB,并将重组质粒转化大肠埃希菌BL21(DE3)感受态细胞。重组子经酶切、PCR鉴定和核酸序列分析正确后,异丙基硫代半乳糖苷(IPTG)诱导重组蛋白的表达,用SDS-PAGE及Western印迹进行鉴定。结果:限制性核酸内切酶酶切鉴定、PCR和核酸序列分析表明,扩增出了淋病奈瑟菌外膜蛋白1 047 bp的PorB基因,成功构建了重组质粒pGEX-4T-PorB,经SDS-PAGE及Western印迹分析显示重组质粒pGEX-4T-PorB在大肠埃希菌中得到了高效融合表达。结论:成功构建pGEX-4T-PorB原核表达载体,并在原核系统中得到了高效表达。     

男性泌尿生殖道感染患者奈瑟菌属菌种的基因检测

目的:检测从男性泌尿生殖道感染患者分离的奈瑟菌属菌种的基因并探讨非淋病奈瑟菌的致病性以及奈瑟菌属菌种感染的实验室诊断方法。方法:用聚合酶链反应及核苷酸序列测定方法,检测男性泌尿生殖道感染患者泌尿生殖道分离的奈瑟菌属菌种的淋病奈瑟菌核酸扩增荧光检测试剂的反应及其染色体上16SrRNA基因、淋病奈瑟菌DNA甲基转移酶基因orf1、隐蔽性质粒基因cppB及外膜蛋白基因nspA。结果:男性泌尿生殖道感染患者泌尿生殖道分离的14株奈瑟菌属菌种经16SrRNA基因检测鉴定为淋病奈瑟菌2株、粘液奈瑟菌3株、灰色奈瑟菌3株、干燥奈瑟菌2株、微黄奈瑟菌2株、嗜乳糖奈瑟菌1株、多糖奈瑟菌1株,其中淋病奈瑟菌核酸扩增荧光检测试剂阳性反应的9株、cppB阳性反应1株、orf1阳性反应5株、nspA阳性反应3株。16SrRNA基因检测与常规细菌学方法鉴定结果比较,两者的符合率85.7%。结论:引起男性泌尿生殖道感染的非淋病奈瑟菌缺乏cppB,大多数菌株缺乏淋病奈瑟菌毒力相关的orf1与nspA并且可在淋病奈瑟菌核酸扩增荧光检测试剂检测中显示阳性反应,从而造成淋病奈瑟菌鉴定与淋病诊断的误诊以及其他奈瑟菌感染的漏诊。在疑似奈瑟菌属菌种感染的实验室检查中联合使用常规细菌学方法与基因检测方法,可有助于提高奈瑟菌属菌种鉴定及其感染诊断的准确率。     

淋病奈瑟菌GyrA基因及ParC基因突变与耐喹诺酮类药物的关系

目的 :探讨淋病奈瑟菌DNA回旋酶A亚单位GyrA基因及拓扑异构酶ⅣC亚单位ParC基因突变与耐喹诺酮类药物之间的关系。　方法 :采用DNA序列测定法对 2 0株无锡地区淋病奈瑟菌临床分离株的GyrA及ParC基因的喹诺酮耐药决定区 (QRDR)相关序列进行测序分析 ,并利用琼脂扩散法检测了该 2 0株淋病奈瑟菌临床分离株对诺氟沙星药物的敏感性。　结果 :2 0株被检淋病奈瑟菌菌株在GyrA基因的两个常见突变位点之一的Ser91处均未发生突变 ,却在另一位点Asp95处则均有突变 ;而 19株被检淋病奈瑟菌菌株中有 16株发生了ParC基因的 86、87、88、91位点突变。　结论 :GyrA基因的Asp95位点突变与ParC基因的Asp86、Ser87、Ser88和Glu91位点突变共同参与了淋病奈瑟菌对喹诺酮类药物产生的耐药。     

男性泌尿生殖道非淋球菌奈瑟菌感染的诊断与治疗研究

目的:探讨非淋球菌奈瑟菌感染对男性泌尿生殖道感染诊断与治疗的影响。方法:采集8例急性尿道炎或慢性前列腺炎患者的尿道分泌物、前列腺按摩液或/和精液标本,分别接种淋球菌培养基、血琼脂培养基、沙保氏琼脂培养基和支原体培养基分离淋病奈瑟菌与其他细菌、真菌、支原体以及Gemenez染色检查衣原体。对分离的革兰阴性双球菌分别进行氧化酶、生化反应、药物敏感试验及淋病奈瑟菌核酸荧光聚合酶链反应(PCR)检测与鉴定。根据药物敏感试验结果选择和使用抗菌药物,对患者以静脉注射或/和口服治疗。结果:从8例患者前列腺按摩液或精液内分离的8株革兰阴性双球菌分别鉴定为粘液奈瑟菌3株、灰色奈瑟菌4株、嗜乳糖奈瑟菌1株,各菌株的隐蔽质粒pJD1基因PCR检测均表现与淋病奈瑟菌相似的阳性反应。男性生殖道分离的非淋球菌奈瑟菌具有多重耐药性,对喹诺酮类药物和磷霉素具有显著高的耐药性。静脉或/和口服给予病原菌敏感的头孢菌素或/和米诺环素治疗7d后,病原学复查无菌和患者症状消失。结论:寄生人体上呼吸道的某些正常菌群奈瑟菌也可以感染男性泌尿生殖器官,引起淋病样尿道炎与慢性前列腺炎。仅仅通过形态、氧化酶试验或/和检测隐蔽质粒基因以及其他淋病奈瑟菌非特异性核苷酸序列的PCR方法,可导致淋病的临床和病原学诊断错误。根据病原学检查结果选择药物静脉注射或/和口服,能够有效治愈非淋病奈瑟菌引起的急性尿道炎与慢性前列腺炎等男性生殖器官感染疾病,病原菌耐药性是影响治疗效果的主要因素。     

淋病奈瑟菌致病的免疫学机制

淋病奈瑟菌作为淋病的病原体,人类是其唯一天然宿主,且普遍易感。淋病奈瑟菌感染后,机体一方面通过募集中性粒细胞等炎症细胞来抵抗其入侵,另一方面也可通过抗原呈递等过程来启动机体适应性免疫应答予以消灭,但淋病奈瑟菌存在一系列免疫逃逸机制,使得感染难以清除。在固有免疫系统中,淋病奈瑟菌可分泌核酸酶分解中性粒细胞外诱捕(NETs)、抑制呼吸爆发来抵抗中性粒细胞的杀伤作用,激活NLRP3炎症小体促进炎症细胞的焦亡,亦可调节巨噬细胞的分化削弱炎症反应,结合H因子来逃避补体介导的杀伤作用。淋病奈瑟菌感染难以产生有效的适应性免疫反应及免疫记忆。淋病奈瑟菌促进转化生长因子β(TGF-β)的产生,从而抑制Th1/Th2细胞介导的适应性免疫反应,可与B细胞表面的CEACAM1结合促进B细胞的凋亡,亦可与T细胞表面的CEACAM1结合抑制辅助性T细胞增生,使得B细胞难以产生高亲和力的特异性抗体。随着淋病奈瑟菌耐药性不断增加,这些免疫学研究对于开发新型疗法及疫苗研制具有重要意义。     

淋病奈瑟菌体外对人精子运动参数的影响

目的 :阐明淋病奈瑟菌体外对人精子的运动参数有无直接影响。　方法 :将淋病奈瑟菌制成活菌悬液 ,在体外与生育男性经上游优化处理的精子孵育。细菌 /精子比为 5 0∶1,分别孵育 0、2、4h ,采用计算机辅助的精子分析系统检测人精子运动参数 (前向性运动百分率、直线速度、曲线速度、平均路径速度、直线性、前向性 )。　结果 :细菌与精子的比例为 5 0∶1,孵育 0、2和 4h后 ,人精子运动参数无明显变化。　结论 :淋病奈瑟菌与精子的比例为5 0∶1,孵育 4h内 ,淋病奈瑟菌体外对人精子运动参数无直接影响     

从淋病病人结膜囊和尿道分泌物中同时分离出淋病奈瑟菌

淋病奈瑟菌是淋病的病原菌 ,很少引起成人的结膜炎 ,最近作者从一结膜炎病人的结膜囊分泌物中分离到一株 ,其培养特性、生化反应、药敏试验与其尿道分泌物分离到的淋病奈瑟菌的完全相同。1　临床资料病人 ,男性 ,4 2岁 ,因结膜炎来本院眼科门诊就诊 ,检查发现结膜充血较明显 ,脓性分泌物较多。实验室检查 :血液ＷＢＣ为 7.6× 10 9/Ｌ ,Ｎ 0 .70 ,Ｌ0 .2 8,Ｍ 0 .0 2。取结膜囊分泌物直接涂片 :脓细胞30～ 4 5个 /ＨＰ ,革兰氏染色为阴性双球菌 ;细菌培养 ,分离出淋病奈瑟菌纯培养。遂令其留取尿道分泌物做细菌培养 ,同样分离出淋病奈瑟菌。…     

南京地区112株淋病奈瑟菌对抗生素的敏感性

淋病是我国目前重点防治的性传播疾病之一 ,在几种性病中发病人数居于首位。临床上治疗淋病多采用抗生素 (口服或肌注 ) ,随着抗生素的广泛应用或滥用以及细菌染色体突变等原因 ,逐渐产生了耐药性淋病奈瑟菌菌株。连续或间断地监测淋病奈瑟菌临床分离株对抗生素的敏感性 ,可了解现行淋病治疗方案的有效性 ,为治疗药物的选择提供参考依据。现将 1999年南京地区 112株淋病奈瑟菌对 5种抗生素的敏感性结果报告如下。1　材料和方法1 1　菌株来源　由全国性病麻风病控制中心性病门诊病人的泌尿生殖道中分离。菌株经菌体形态、菌落形态及氧化酶试…     

五苦栓杀精抑菌作用的实验研究

目的 :了解中药“五苦栓”杀精、抑菌作用。　方法 :运用中药五倍子、苦参粗提物配制成杀精栓剂 (WK) ,对大鼠精子行杀精、生育、阴道粘膜刺激及体外抑菌试验。　结果 :WK具有较明显杀精作用 ,其体外杀精效果在高浓度组超过NP 9阳性对照组 (P <0 .0 1) ,中等浓度时与NP 9无显著性差异 (P >0 .0 5 )。体内杀精效果与NP 9无差异 (P >0 .0 5 )。对淋病奈瑟菌及霉菌的抑制作用强于NP 9(P <0 .0 1) ,且安全性实验未发现有明显副作用。　结论 :“五苦栓”杀精及体外抑制淋病奈瑟菌、霉菌作用确切 ,具有临床开发价值。     

男性性病病人支原体、衣原体及淋病奈瑟菌感染分析

性病病人解脲支原体(Ureaplasma urealyticum,Uu)、沙眼衣原体(Chlamydia trachomatis,Ct)和淋病奈瑟菌(Neisseriagonorrhoeae,Ng)3种病原体感染研究已有较多的报道.     

Current status of vaccine development in sexually transmissible diseases

A prophylactic vaccine represents a major hope for the control of sexually transmitted diseases. The current general vaccine strategies and the status of vaccine development against infections with Neisseria gonorrhoeae, Treponema pallidum, Chlamydia trachomatis and Herpes simplex virus are described. Vaccines consisting of whole infectious agents are replaced by protective subunits. A subunit vaccine has the advantage to be free from other components, which are not relevant for protection and which may confer unwanted side effects. At the present time vaccine development against infections with Neisseria gonorrhoeae and Herpes simplex virus seems to be the most progressed. With monoclonal antibodies several surface components could be identified, which are of importance for the pathomechanism. With Treponema pallidum and Chlamydia trachomatis the development is delayed by unsolved problems of immunity. For the production of vaccines molecular-biologic methods, like protein synthesis or gene-cloning will be used. Genetically modified live vaccines or polytope hybrid vaccines will gain importance in the future.     

In vitro activity of a new 'higher-lactam' antibacterial agent LY 193239

In vitro activity of the new bicyclic pyrazolidinone LY 193239 (Eli Lilly) was evaluated against 52 clinical isolates of Haemophilus influenzae (4 were beta-lactamase producers), 32 Enterococcus faecalis, 14 Neisseria gonorrhoeae (1 beta-lactamase-positive) and 19 Neisseria meningitidis. Activity was best against Neisseria spp. and H. influenzae, including penicillinase-producing strains. Results of the time-kill study against a non-enzyme-mediated penicillin resistant strain of N. meningitidis indicate that exposure to an antibacterial concentration four times the minimal inhibitory concentration was bactericidal. E. faecalis was insensitive.     

阿奇霉素治疗慢性淋菌性前列腺炎疗效观察

目的 :观察门冬氨酸阿奇霉素注射剂对慢性淋菌性前列腺炎的疗效。　方法 :以阿奇霉素 1次 /d静脉单剂给药治疗轻到中度的淋病奈瑟菌所致的慢性淋菌性前列腺炎 19例 ,观察其疗效及副作用。　结果 :12例治愈(6 3 16 % ) ,5例显效 ,总有效率达 89 4 7%。　结论 :用阿奇霉素注射剂短期治疗慢性淋菌性前列腺炎安全有效 ,并可消除依从性的问题     

Single low-dose ceftriaxone for the treatment of gonococcal ophthalmia--implications for the national programme for the syndromic management of sexually transmitted diseases.

We prospectively analysed a total of 21 baby-mother pairs with culture-proven Neisseria gonorrhoeae treated with a single low dose of ceftriaxone, namely 62.5 mg for babies and 125 mg for mothers respectively. N. gonorrhoeae was eradicated from all babies' eyes with no residual damage, as well as from the mothers' cervixes. A single low dose of 62.5 mg ceftriaxone has emerged as the treatment of choice for gonococcal ophthalmia neonatorum because of its excellent activity against N. gonorrhocae, including penicillinase-producing strains.     

Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro

Aim: The effects of certain uropathogenic microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermidis and Mycobacterium tuberculosis) on human sperm motility characteristics were studied in vitro. Methods: In 10 healthy fertile men, ejaculates were aseptically obtained by masturbation and With a swim-up technique, a sperm suspension of high motility and purity was obtained. Several uropathogenic bacteria were obtained from outpatients with genitourinary tract infections. The sperm suspension was incubated with the pathogens at a bacteria: sperm ratio of 50:1 at 37℃. The sperm mobility parameters were estimated with a computerassisted sperm analyzer (CASA) provided with a multiple-exposure photography system (Madi Corp., Zhejiang, China). Measurements were carried out at 0, 2 and 4 hours of incubation. Results: Staphylococcus aureus significantly decreased the sperm motility and viability, but Staphylococcus epidermidis, Mycobacterium tuberculosis and Neisseria gonorrhoeae d     

Epidemiological survey of sexually transmitted male urethritis in Kyoto Prefecture

The data of sexually transmitted urethritis in males have been collected at 24 institutes in Kyoto Prefecture since October, 2002. The data collected from January to December in 2004 are summarized herein. A total of 1,275 patients were diagnosed with urethritis during this period. Microbiological examinations isolated Neisseria gonorrhoeae alone in 368 (29%), Chlamydia tracomatis alone in 336 (26%), both in 85 (7%), and others in 453 (36%). Male patients under 20 years old tended to have Chlamydial urethritis, alone or combined with gonococcal infection, and had a predominant infectious source, a non-commercial-sexual-worker female partner, suggesting a profound problem in sexual life of adolescents. The urologist preferred to use quinolones as the first therapeutic modality against male urethritis. However, drug resistance of N. gonorrhoeae, especially against quinolones, has rapidly progressed, which was also observed by a sensitivity examination test. Antibiotics should be used adequately against male urethrits according to the recent guidelines.     

Sexually transmitted pathogens in acute pelvic inflammatory disease

The prevalence of sexually transmitted pathogens in two groups of women was studied: 50 women with clinical diagnoses of acute pelvic inflammatory disease (PID) and 50 asymptomatic women attending a family planning clinic (FPC). Genital specimens, collected by non-invasive procedures, were examined. Endocervical Neisseria gonorrhoeae was present in 62% of the PID group and 10% of the FPC group (P less than 0.0001). One-third of N. gonorrhoeae isolates were penicillinase-producing strains. Chlamydia trachomatis was isolated from the endocervix in 30% of the PID group and 26% of the FPC group (P = 0.8240 NS). Mycoplasma hominis was more prevalent in the vaginas and endocervices of the PID group than the FPC group (84% and 72% v. 50% and 42%; P = 0.0006 and 0.0047 respectively). Trichomonas vaginalis was present in 56% of the PID group and 20% of the FPC group (P = 0.0004). Syphilis serology was positive in 34% of the PID group and 10% of the FPC group (P = 0.0026). In the PID group of patients, 8% were positive for HBsAg. Antibody to the human immunodeficiency virus was not detected in any of the 100 women. The high prevalence of recognised sexually transmitted pathogens underlines the need for appropriate antimicrobial agent(s) active against N. gonorrhoeae, C. trachomatis and M. hominis in patients with PID. In view of the high prevalence of penicillinase-producing strains of N. gonorrhoeae, routine use of an antibiotic active against such strains is desirable.     

A microbiological study of failed penicillin therapy for gonococcal urethritis in Durban

Seventy-five men presenting with persistent urethral discharge after penicillin therapy were investigated for sexually transmitted pathogens during July - September 1987. The major aetiological agent isolated was Neisseria gonorrhoeae (58 patients (77.3%]. Penicillinase-producing N. gonorrhoeae (PPNG) accounted for 58.6% of 58 isolates. Chlamydia trachomatis was detected in 10.7% of patients and Trichomonas vaginalis in 14.7% of patients. When there is a high prevalence of PPNG, the use of penicillin as a first-line agent for therapy should be discontinued in favour of an agent active against PPNG and non-PPNG strains. Furthermore, in view of the relatively high prevalence of T. vaginalis, patients returning with persistent urethral discharge should be investigated and treated for infection with this protozoan.