淋病奈瑟球菌的耐药性检测

目的了解淋病奈瑟球菌对临床常见药物的耐药性。方法采用琼脂稀释法测定13种抗菌药物对35株淋病奈瑟球菌的MICs;Nitrocefin(头孢硝噻酚)纸片检测β-内酰胺酶,并采用PCR测定其基因型。结果淋病奈瑟球菌对青霉素、四环素、环丙沙星与头孢呋辛的耐药率分别为65．7％、60．0％、88．6％与14．3％,对第3、4代头孢菌素、大观霉素非常敏感,耐药率均为0％;42．9％的菌株产生TEM型β-内酰胺酶。结论淋病奈瑟球菌对青霉素、四环素类和氟喹诺酮类的耐药性高,治疗宜选用第3、4代头孢菌素和大观霉素等,同时加强耐药性监测。     

淋病奈瑟菌感染实验室检测技术的进展

细菌培养是诊断淋病奈瑟菌(NG)感染的"金标准",也是目前诊断淋病最可靠的方法.但淋病奈瑟菌在人体外抵抗力极弱,不易培养;仅做细菌形态学检查,诊断意义有限.随着淋病奈瑟菌耐药性不断增强,多重耐药菌出现并广泛传播.高敏感性、高特异性、操作简便的分子生物学检验技术,如单管多重聚合酶链反应(PCR)技术等,已开始用于淋病奈瑟...     

淋球菌耐药性与耐药质粒的研究

为了解湛江地区淋球菌耐药现状及耐药质粒的分布,并初步探讨其相互关系,对１９９８￣１９９９年广东省湛江地区健康鉴定出的９８株淋球菌流行株,应用纸片扩散法测定细胞对１０种抗生素的敏感性,并采用碱裂解法进行质粒抽提,分析耐药质粒的分布情况,选取ＮＧ４、ＮＧ３１、ＮＧ４３、ＮＧ７０进行细菌的接合试验,以观察耐药质粒能否通过接合进行传递以及传递后受体菌耐药性的变化。结果显示６．１２％菌株对全部抗生素敏感,４８．９６％的菌株对３种及３种以上的抗生素耐药;共检出四种不同分子量的质闰,７．４ｋｂ和４．２ｋｂ质粒检出率较高,分别为５９．１６％和６７．３２％,质粒谱型１２种,以７．４ｋｇ＋４．２ｋｇ和３９．５ｋｇ＋７．４ｋｇ＋４．２ｋｇ为主,占３８．７６％。在细菌的接合传递试验中,ＮＧ４３可通过接合传递将其耐药性传递给淋球菌及大肠     

Pseudo-transposition of a Tn5 derivative in Neisseria gonorrhoeae

Abstract We constructed a Tn5 derivative for potential use in transposon mutagenesis of Neisseria gonorrhoeae . It was incorporated into the chromosome apparently at random following transformation, but the insertion events were dependent on a functional RecA and independent of a functional transposase. Furthermore, in most cases there was an incomplete transposon inserted with little or no IS50 insertion sequence. These observations suggest that TnJ transposition may not be possible in N. gonorrhoeae and that this organism may have an unexplored illegitimate recombination system.     

Neisseria gonorrhoeae possesses two nicotinamide adenine dinucleotide-independent lactate dehydrogenases

Abstract An important metabolic capability of Neisseria gonorrhoeae is the utilization of host-derived lactate. Two isoenzymes of the membrane-associated, pyridine dinucleotide-independent type of lactate dehydrogenase (iLDH) participate in lactate assimilation, but exhibit distinctive properties. Isoenzyme iLDH-I utilized lactate exclusively as substrate, exhibiting a preference for the D-isomer. In contrast, isoenzyme iLDH-II exhibited broad substrate specificity (lactate, phenyllactate, and 4-hydroxyphenyllactate), but was stereospecific for the L-isomers. These results explain the difficulty in isolating mutants unable to utilize lactate.     

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.     

淋病奈瑟菌对hCD46基因转染COS-1细胞的黏附

为研究hCD46在菌毛介导的淋病奈瑟菌宿主特异性黏附过程中的作用。用连接PCR技术扩增出启动子与cDNA相连的hCD46小基因,并将其置换出pcDNA3.1载体中的CMV启动子,构建成重组真核表达载体pCD46。转染COS-1细胞后用间接免疫荧光法检测到hCD46 cDNA在其自身启动子的指导下可在COS-1细胞膜表面有效表达,Western blotting检测表明表达产物的分子正确,用流式细胞术分选表达hCD46的基因转染细胞COS-1-46。细菌黏附实验显示菌毛阳性淋病奈瑟菌临床分离株对COS-1-46细胞有较强的黏附性。提示hCD46是菌毛介导的淋病奈瑟菌特异性黏附于人黏膜细胞的一种重要受体,hCD46小基因可用于淋病奈瑟菌感染转基因小鼠模型的制备。     

淋球菌AFLP基因分型的初步研究

采用扩增片段长度多态性技术(AFLP)对奈瑟氏淋球菌菌株进行基因分型研究。以EcoRI和MesI酶切26株淋球菌临床分离株基因组,并进行AFLP分析。同一地区的淋球菌分离株之间存在相当大的DNA多态性。AFLP是鉴别淋球菌临床分离株有用而敏感的基因分型技术,有助于了解流行淋球菌菌株的来源、流行菌株之间的克隆相关性,以及抗生素耐药性菌株的传播情况。     

有核细胞对淋球菌存活力的影响

目的观察不同浓度的淋球菌在有核细胞内外和体外的存活状况。方法白带标本在密闭保湿和开放干燥的环境下,分别在0、12、24、48h接种于巧克力平板,另将淋球菌用生理盐水配成不同浓度的菌液与有核细胞悬液混合培养30min、1h、5h,然后分别取不同时间段的混合培养液接种于巧克力平板。结果在密闭保湿状况下,白带标本中的淋球菌在中量时存活时间长,少量或大量时其存活力相对较低;在干燥状况下,淋球菌存活时间短;在菌量很少（10／ml）时,有核细胞可以吞噬淋球菌而导致淋球菌存活时间减少,但在菌量较多时（大于300／ml）时,有核细胞可以延长淋球菌存活时间。结论温暖湿润环境有利淋球菌存活;有核细胞对淋球菌既具有保护作用又有杀伤作用。     

DNA methylase activities in a Neisseria gonorrhoeae extract

Abstract Resistance of Neisseria gonorrhoeae DNA to cleavage by various restriction endonucleases suggests the existence of modification enzymes which protect the corresponding recognition sequences. We indeed found methylase activities in N. gonorrhoeae extracts. These activities lead to the methylation of adenine and cytosine residues in bacteriophage λ DNA and DNA from an Escherichia coli Dam ⁻ strain. They also result in partial protection of λ DNA to cleavage by the restriction endonucleases Hae II, Hae III, Bam HI and, Sac II.     

2010年广州地区淋球菌耐药监测结果分析

目的了解广州地区淋球菌对抗生素耐药性的变化及PPNG和TRNG的流行趋势。方法用琼脂稀释法测定头孢曲松、大观霉素、环丙沙星、阿奇霉素和四环素的最低抑菌浓度（MIC）;用纸片碘量法检测β-内酰胺酶。结果83株淋球菌检出PPNG24株（28．9％）、TRNG50株（60．2％）、环丙沙星耐药率高达98．8％,高度耐药株（MIC≥16mg／L）43株（51．8％）,而76株淋球菌中阿奇霉素耐药株11株（14．5％）,均未出现对头孢曲松、大观霉素耐药的菌株,抗菌活性强。结论合理规范使用抗生素及动态监测淋球菌耐药性变迁是临床减少淋球菌耐药菌株出现的有效办法。     

Competition between Neisseria gonorrhoeae and Staphylococcus epidermidis during iron-limited or replete continuous culture

Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.     

利用雌激素构建小鼠淋病奈瑟菌感染模型

目的探讨用雌激素构建淋病奈瑟菌感染小鼠模型的方法。方法淋病奈瑟菌WHO-A接种经皮下注射苯甲酸雌二醇和真皮下埋植尼尔雌醇片（雌三醇）预处理的雌性ICR小鼠,小鼠阴道拭子接种T-M选择培养基培养分离淋病奈瑟菌,取小鼠阴道分泌物涂片染色观察淋病奈瑟菌感染情况,比较两种雌激素对小鼠淋病奈瑟菌感染的差异。结果与对照组相比,苯甲酸雌二醇小鼠体内淋病奈瑟菌有短时间的定植,而尼尔雌醇组与对照组无差异。结论雌激素（尤其是雌二醇）有利于淋病奈瑟菌在小鼠阴道内的定植。     

Neisseria gonorrhoeae survives within and modulates apoptosis and inflammatory cytokine production of human macrophages

The human‐adapted organism Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection. It readily colonizes the genital, rectal and nasalpharyngeal mucosa during infection. While it is well established that N. gonorrhoeae recruits and modulates the functions of polymorphonuclear leukocytes during infection, how N. gonorrhoeae interacts with macrophages present in infected tissue is not fully defined. We studied the interactions of N. gonorrhoeae with two human monocytic cell lines, THP‐1 and U937, and primary monocytes, all differentiated into macrophages. Most engulfed bacteria were killed in the phagolysosome, but a subset of bacteria was able to survive and replicate inside the macrophages suggesting that those cells may be an unexplored cellular reservoir for N. gonorrhoeae during infection. N. gonorrhoeae was able to modulate macrophage apoptosis: N. gonorrhoeae induced apoptosis in THP‐1 cells whereas it inhibited induced apoptosis in U937 cells and primary human macrophages. Furthermore, N. gonorrhoeae induced expression of inflammatory cytokines in macrophages, suggesting a role for macrophages in recruiting polymorphonuclear leukocytes to the site of infection. These results indicate macrophages may serve as a significant replicative niche for N. gonorrhoeae and play an important role in gonorrheal pathogenesis.     

淋病流行株多重耐药性与mtrRCDE外排系统的关系

为了探讨mtrRCDE外排系统与淋病流行株多重耐药性的关系 ,应用K B法和琼脂稀释法从湛江地区分离出 6 2株淋球菌多重耐药株。利用煮沸法提取细菌DNA ,PCR扩增mtrR基因 ,并对扩增产物测序 ,比较敏感株与多重耐药株的差异。结果显示 :5株敏感株和 2株耐青霉素淋球菌无mtrR的突变 ,10株多重耐药株均有mtrR基因的突变 ,表现为第 12 4位和第 139位G→A。从而提示 ,mtrRCDE外排系统与淋病流行株的多重耐药性密切相关。     

淋病奈瑟菌感染的实验室诊断技术概述

淋病奈瑟菌是淋病的病原菌,淋病不及时诊断和治疗会引起严重的并发症,早期诊断和治疗是控制淋病传播的关键。试验诊断技术在淋病的诊断和治疗中具有十分重要的作用。目前,常用的试验诊断方法有涂片镜检、分离培养、免疫学方法、分子生物学检查和快速检查等。分泌物直接涂片染色镜检大多常用革兰染色,但是有其局限性,细菌培养是诊断淋病的金标准,也是目前诊断淋病最可靠的方法,但淋病奈瑟菌培养困难,仅做细菌学培养检查,诊断意义有限。随着新仪器的研发以及现代分子生物学理论和技术的发展,快速、准确、特异、敏感的分子生物学技术已经成为试验诊断的重要手段,具有广阔的应用前景,临床上还需要开发新的核酸扩增试验技术,以便更好地指导临床治疗。临床医生在实际诊疗过程中应该根据患者的实际情况正确选择检测方法,有时建议选择两种以上的检测方法以提高阳性检出率。本文就淋病奈瑟菌感染的试验诊断技术的研究进展加以概述。     

A fully defined, clear and protein-free liquid medium permitting dense growth of Neisseria gonorrhoeae from very low inocula

Neisseria gonorrhoeae is difficult to cultivate in liquid medium. Currently there are no liquid media, defined or undefined, that reliably permit growth of this bacterium from low inocula. Standard clinical laboratory broths may allow multiplication of some strains of gonococci from large inocula, but such media incorporate infusates, extracts or digests and are therefore undefined. In this study, 20 gonococci of ten auxotypes were tested in various experimental media in the development of an easily prepared chemically defined, clear and protein-free liquid medium. The final medium - GW medium - allowed the growth of three clinical isolates of gonococci from inocula of <10(3) CFU mL(-1) to >10(8) CFU mL(-1) by 24 h. None of four commercially-available broths (nutrient broth, brain heart infusion, tryptone soya broth, and Mueller-Hinton broth) tested in parallel reliably supported growth of these isolates to the same extent. GW medium should be useful for studies of the growth of gonococci under different conditions and, as the medium is clear and colorless, this can be monitored turbidometrically. GW medium may be suitable as a basal medium for biochemical identification tests, antimicrobial susceptibility determinations and antimicrobial synergy studies.