甲硝唑治疗细菌性阴道病前后阴道菌群的结构变化及其预后相关性

目的研究经甲硝唑规范治疗的细菌性阴道病患者在用药后6~8d随访时的菌群结构与治疗前以及治疗结局之间的关系,探讨治愈人群与治疗失败人群在6~8d这一观察点的阴道菌群多样性的差异,从而为实现细菌性阴道病治疗结局的预测提供理论依据。方法选取2012年9月至2013年7月在北京大学第一医院妇产科门诊诊断为细菌性阴道病的育龄期女性为研究对象,规范使用甲硝唑凝胶治疗,分别采集患者用药前、用药后6~8d、用药后1个月的阴道分泌物,提取细菌组DNA,进行454焦磷酸测序,分析阴道微生物菌群的多样性和丰度差异,探讨细菌性阴道病患者阴道菌群的构成差异与预后之间的相关性。结果共61名患者完成了2次随访,共计183份阴道分泌物样品。对所有采集的标本进行454焦磷酸测序分析,共发现192种细菌。用药后6~8d的革兰染色涂片结果显示所有患者的阴道菌群镜下可分为三种情况:正常菌群、菌群抑制、异常菌群。镜检涂片为正常菌群的治愈率最高,其次是菌群抑制,异常菌群的治愈率最低。454焦磷酸测序分析显示甲硝唑治疗前后菌群的多样性和丰度发生了一定程度的变化,乳杆菌和球菌的含量未受明显影响,而细菌性阴道病致病菌的含量明显减少。结论甲硝唑可以有效减少细菌性阴道病致病菌的数量而不影响乳杆菌的含量,但是对以球菌为主的患者治疗效果差。对高通量测序结果和革兰染色涂片结果的综合分析显示甲硝唑用药后6~8d的阴道菌群对细菌性阴道病1个月后的治疗结局具有预测价值。     

甲硝唑治疗细菌性阴道病前后阴道菌群的 结构变化及其预后相关性

摘要：目的 研究经甲硝唑规范治疗的细菌性阴道病患者在用药后6~8 d随访时的菌群结构与治疗前以及治疗结局之间的关系,探讨治愈人群与治疗失败人群在6~8 d这一观察点的阴道菌群多样性的差异,从而为实现细菌性阴道病治疗结局的预测提供理论依据。方法 选取2012年9月至2013年7月在北京大学第一医院妇产科门诊诊断为细菌性阴道病的育龄期女性为研究对象,规范使用甲硝唑凝胶治疗,分别采集患者用药前、用药后6~8 d、用药后1个月的阴道分泌物,提取细菌组DNA,进行454焦磷酸测序,分析阴道微生物菌群的多样性和丰度差异,探讨细菌性阴道病患者阴道菌群的构成差异与预后之间的相关性。结果 共61名患者完成了2次随访,共计183份阴道分泌物样品。对所有采集的标本进行454 焦磷酸测序分析,共发现192种细菌。用药后6~8 d的革兰氏染色涂片结果显示所有患者的阴道菌群镜下可分为三种情况：正常菌群、菌群抑制、异常菌群。镜检涂片为正常菌群的治愈率最高,其次是菌群抑制,异常菌群的治愈率最低。454焦磷酸测序分析显示甲硝唑治疗前后菌群的多样性和丰度发生了一定程度的变化,乳杆菌和球菌的含量未受明显影响,而细菌性阴道病致病菌的含量明显减少。结论 甲硝唑可以有效减少细菌性阴道病致病菌的数量而不影响乳杆菌的含量,但是对以球菌为主的患者治疗效果差。对高通量测序结果和革兰氏染色涂片结果的综合分析显示甲硝唑用药后6~8 d的阴道菌群对细菌性阴道病1个月后的治疗结局具有预测价值。     

基于实时定量PCR技术的育龄期细菌性阴道病患者 阴道菌群结构分析及其临床应用

摘要：目的 探讨育龄期女性细菌性阴道病（bacterial vaginosis,BV）患者阴道优势菌群变化及其与临床指标的相关性。方法 选择本院门诊确诊的育龄期BV患者35例和同期入院体检的健康体检女性37例,无菌拭子采集阴道中段壁分泌物,提取细菌基因组DNA,采用实时定量PCR技术（real-time quantitative PCR,qPCR）进行阴道优势细菌检测,并进行其与临床指标如阴道pH和Nugent评分相关性分析。结果 乳杆菌属细菌及其种水平的细菌如惰性乳杆菌、卷曲乳杆菌、詹氏乳杆菌等在BV患者中均显著下降,BV相关阴道致病细菌如加德纳菌属、奇异菌属、埃格特菌属、巨型球菌I型菌属、纤毛菌属和普氏菌属显著升高（P<0.05）。阴道致病菌群与阴道pH和Nugent评分呈显著正相关,而阴道卷曲乳杆菌和惰性乳杆菌与其呈负相关。结论 育龄期BV患者阴道优势菌群显著失衡,并与阴道pH和Nugent评分显著相关,提示阴道优势菌群改变参与BV发生发展。     

不同人群阴道乳杆菌分离比较的研究

目的比较细菌性阴道病患者、抗生素治疗后BV患者和健康妇女阴道内乳杆菌的分布差异。方法对124例(包括40例细菌性阴道病患者和23例抗生素治疗后细菌性阴道病患者)年龄为19~65岁的妇女阴道分泌物进行乳杆菌及产H2O2乳杆菌的分离和半定量计数。结果健康妇女阴道内乳杆菌检出率和活菌数量明显高于细菌性阴道病患者和抗生素治疗后细菌性阴道病患者(P<0.01);产H2O2乳杆菌是健康妇女阴道内优势菌,其检出率明显高于细菌性阴道病患者。结论阴道内乳杆菌特别是产H2O2乳杆菌是健康妇女阴道乳杆菌的重要成员,在防治细菌性阴道病发生中起重要作用,且对多种抗生素敏感。     

甲硝唑与乳杆菌活菌制剂对细菌性阴道病患者阴道微生物群影响的对比性研究

目的对300例细菌性阴道病（Bacterial Vaginosis,BV）患者随机使用甲硝唑（100例）、乳杆菌活菌制剂定君生（100例）及甲硝唑联合定君生（100例）,观察乳杆菌在阴道内定植情况及用药前后阴道内微生物多样性的变化,为临床治疗细菌性阴道病提供实验依据。方法分别提取300例BV患者用药前和用药后的阴道微生物总基因组DNA,以德氏乳杆菌16SrDNA特异性引物进行PCR,检测患者用药后德氏乳杆菌在阴道内的定植情况。以细菌16SrRNAV3区通用引物进行PCR,扩增产物进行变性梯度凝胶电泳（DGGE）,对所得指纹图谱进行分析,研究用药对微生态变化的影响状况。结果甲硝唑治疗组用药前后均没有德氏乳杆菌检出;定君生治疗组用药后德氏乳杆菌检出率为54％;甲硝唑联合定君生治疗组用药后德氏乳杆菌检出率为76％。甲硝唑治疗组用药后微生物多样性显著降低,微生物群结构失衡;定君生治疗组用药后微生物多样性有所降低,微生物群结构无显著差异;甲硝唑联合定君生治疗组用药后微生物多样性降低,但较前两组有所增加,微生物群结构差异无统计学意义。结论甲硝唑显著降低BV患者阴道微生物多样性,引起微生态失衡;定君生对BV患者阴道微生态的影响明显小于甲硝唑;定君生的使用有利于甲硝唑f预后阴道微生物多样性的恢复和微生态平衡的重建。     

乳酸杆菌活菌制剂用于治疗老年性阴道炎合并细菌性阴道病临床探讨

目的探讨乳酸杆菌活菌制剂用于治疗老年性阴道炎合并细菌性阴道病的临床疗效。方法对118例老年性阴道炎合并细菌性阴道病患者随机分3组,分别予以乳酸杆菌活菌制剂局部用药和（或）口服甲硝唑,进行疗效比较。结果乳酸杆菌活菌制剂和甲硝唑联合应用对治疗老年性阴道炎合并细菌性阴道病疗效显著高于单一用药的疗效,组间差异具有显著性（P〈0.05）;复发率低于单一用药。结论乳酸杆菌活菌制剂与甲硝唑联合治疗老年性阴道炎合并细菌性阴道病有比较好的治疗效果,不易复发。     

联合用药治疗细菌性阴道病

根据细菌性阴道病的特点,可以选择用微生物类药品联合硝基咪唑类药品治疗细菌性阴道病（BV）,主要是为了寻找一种疗效好且不易复发的方法。药敏试验发现,定君生（乳杆菌活菌胶囊,一种微生物类药）对甲硝唑（硝基咪唑类药）极不敏感,两者可以联合用药。近一年来,通过对门诊50例细菌性阴道病患者的联合用药治疗,取得了满意的效果,可以达到这一要求。     

乳杆菌活菌胶囊预防细菌性阴道病复发的临床效果

目的 评价乳杆菌活菌胶囊(商品名:定君生)预防细菌性阴道病复发的临床效果.方法 将2011年10月至2012年5月深圳市南山区妇幼保健院妇科门诊140例细菌性阴道病患者随机分为2组,研究组在细菌性阴道病患者治愈后给予乳杆菌活菌胶囊巩固治疗,阴道上药,1粒/(次·d),连用10 d;对照组仅给予常规治疗.结果 研究组复发率明显低于对照组,差异具有统计学意义(P＜0.05).结论 乳杆菌活菌胶囊能改善及重建阴道微生态平衡,从而有效减少细菌性阴道病的复发.     

乳杆菌活菌胶囊联合甲硝唑栓治疗细菌性阴道病的疗效及预防复发作用

目的探讨乳杆菌活菌胶囊联合甲硝唑栓治疗细菌性阴道病的疗效及预防复发作用。方法将88例细菌性阴道病患者的随机分为观察组和对照组。观察组患者予以乳杆菌活菌胶囊和甲硝唑栓联合治疗,对照组患者予以单纯的甲硝唑栓治疗。乳杆菌活菌胶囊阴道放置,每次1粒,每晚1次,连用10d。甲硝唑栓阴道放置,阴道放置,每次1粒,每晚1次,连用10d。两组患者治疗期间及复查前禁止性生活,避免冲洗阴道及使用抗生素和微生态制剂。观察两组患者治疗停药后2周的临床疗效,并比较随访3个月及6个月内的复发率。结果治疗停药后2周,观察组的临床总有效率为95．45％,明显高于对照组的81．82％（χ2=4．06,P〈0．05）。治疗后进行随访,观察组的3个月及6个月内复发率分别为7．14％和16．67％,均明显低于对照组的25．00％和38．89％（χ2=4．75和4．87,P〈0．05）。结论乳杆菌活菌胶囊联合甲硝唑栓治疗细菌性阴道病具有较好的疗效,并降低其复发率,具有治疗与预防细菌性阴道病的双重作用。     

细菌性阴道病及其 合并盆腔炎患者的阴道菌群分析

目的对比细菌性阴道病及其合并盆腔炎患者与健康个体的阴道菌群,分析阴道菌群的结构,为确定该疾病的特征细菌及研究致病机制奠定基础。方法采用临床Amsel标准筛选的17例细菌性阴道病患者、13例细菌性阴道病合并盆腔炎患者和52例健康者,使用无菌拭子采集阴道后穹窿分泌物以提取细菌基因组DNA。采用PCR技术扩增上一步得到的16S rRNA片段,而后将扩增产物通过变形梯度凝胶电泳(DGGE)分离以得到阴道细菌种属结构图谱,应用Quantity One软件进行聚类分析,应用凝胶测序法进行特异条带分析。检测16S rRNA基因,具体研究样品中的物种分类。结果细菌性阴道病及其合并盆腔炎患者的阴道菌群的构成与健康者相比较具有显著性差异。其中,Firmicutes等菌门细菌减少,Actinobacteria等菌门细菌增多;G.vaginallis、P.vaginallis等厌氧菌数量均增加,L.crispatus、L.iners等益生菌数量均有减少。结论细菌性阴道病及其合并盆腔炎患者的生殖道内的合并感染改变了阴道内的原有微生态平衡。两组疾病组患者的阴道菌群构成相比于健康对照组变化明显。     

Contribution of bacterial outer membrane vesicles to innate bacterial defense

Background

Microorganisms produce cell-wall-degrading enzymes as part of their strategies for plant invasion/nutrition. Among these, pectin lyases (PNLs) catalyze the depolymerization of esterified pectin by a β-elimination mechanism. PNLs are grouped together with pectate lyases (PL) in Family 1 of the polysaccharide lyases, as they share a conserved structure in a parallel β-helix. The best-characterized fungal pectin lyases are obtained from saprophytic/opportunistic fungi in the genera Aspergillus and Penicillium and from some pathogens such as Colletotrichum gloeosporioides. The organism used in the present study, Colletotrichum lindemuthianum, is a phytopathogenic fungus that can be subdivided into different physiological races with different capacities to infect its host, Phaseolus vulgaris. These include the non-pathogenic and pathogenic strains known as races 0 and 1472, respectively.

Results

Here we report the isolation and sequence analysis of the Clpnl2 gene, which encodes the pectin lyase 2 of C. lindemuthianum, and its expression in pathogenic and non-pathogenic races of C. lindemuthianum grown on different carbon sources. In addition, we performed a phylogenetic analysis of the deduced amino acid sequence of Clpnl2 based on reported sequences of PNLs from other sources and compared the three-dimensional structure of Clpnl2, as predicted by homology modeling, with those of other organisms. Both analyses revealed an early separation of bacterial pectin lyases from those found in fungi and oomycetes. Furthermore, two groups could be distinguished among the enzymes from fungi and oomycetes: one comprising enzymes from mostly saprophytic/opportunistic fungi and the other formed mainly by enzymes from pathogenic fungi and oomycetes. Clpnl2 was found in the latter group and was grouped together with the pectin lyase from C. gloeosporioides.

Conclusions

The Clpnl2 gene of C. lindemuthianum shares the characteristic elements of genes coding for pectin lyases. A time-course analysis revealed significant differences between the two fungal races in terms of the expression of Clpnl2 encoding for pectin lyase 2. According to the results, pectin lyases from bacteria and fungi separated early during evolution. Likewise, the enzymes from fungi and oomycetes diverged in accordance with their differing lifestyles. It is possible that the diversity and nature of the assimilatory carbon substrates processed by these organisms played a determinant role in this phenomenon.     

A bacterial actin unites to divide bacterial cells

EMBO J 31 10, 2249–2260 (2012); published online March302012Once thought to exist only in eukaryotic cells, the highly conserved bacterial cytoskeleton is now known to function analogously to its eukaryotic counterparts, particularly in cell shape and division. For instance, the actin-like MreB protein and its homologs are important to maintain cell shape in many rod-shaped bacteria, probably by organizing how peptidoglycan is synthesized. FtsZ, a tubulin homolog, forms a scaffold for the cytokinetic ring, or divisome, by GTP-dependent polymerization into protofilaments. In this issue of The EMBO Journal, Szwedziak et al (2012) reveal the first crystal structures of cell division protein FtsA polymerizing into actin-like filaments, along with in vivo evidence that this self-interaction is crucial for proper cell division.FtsA is an actin homolog required for cytokinesis in many bacterial species and has several key roles in cell division, including helping to tether FtsZ to the cytoplasmic membrane via a membrane-targeting sequence (MTS), recruiting other essential proteins to the divisome, and perhaps promoting divisome constriction (de Boer, 2010). Szwedziak et al (2012) recapitulate the FtsZ-FtsA-membrane association in vitro using liposomes with FtsZ and FtsA proteins from Thermotoga maritima. To get a closer look at the FtsA-FtsZ interface, the authors co-crystallize FtsA with the carboxy-terminal tail of FtsZ, which is known to interact with FtsA. Intriguingly, the crystal reveals an FtsA homodimer. Contrary to the previous bioinformatics model of FtsA self-interaction that proposed a 180° rotation between the two subunits (Carettoni et al, 2003), the FtsA-FtsA interface in the crystal structure shows no rotation, similar to F-actin. Szwedziak et al (2012) also show that FtsA can form longer, actin-like polymers in the presence of non-hydrolysable ATP or on lipid monolayers. These results are surprising because FtsA has a divergent subdomain architecture compared to other actin-family proteins (van den Ent and Löwe).A critical question now is whether FtsA needs to form polymers in vivo to function properly. Purified Streptococcus pneumoniae FtsA assembles into large polymers that are not like F-actin, and it remains unclear if these structures are relevant in vivo (Krupka et al, 2012). Wild-type FtsA proteins do not form detectable filaments in cells, but C-terminal truncations of FtsA that remove the MTS form polymers quite readily in cells when overproduced, although they are not functional (Pichoff and Lutkenhaus, 2007). Even so, starting with an MTS truncation derivative of FtsA to visualize in vivo polymers, Szwedziak et al (2012) design site-directed mutants of Bacillus subtilis FtsA based on the FtsA-FtsA interface of their crystals; these fail to assemble into polymers in vivo. Using a similar MTS truncation derivative, Pichoff et al (2012) created random mutations in Escherichia coli FtsA, and found that those mapping to the same interface found by Szwedziak et al (2012) also disrupted polymer formation. Together, these data suggest that these residues are needed for FtsA self-interaction. Perplexingly, when these mutants were subsequently tested for functionality in the context of full-length FtsA, the results were mixed. Pichoff et al (2012) showed that FtsA mutants deficient for self-interaction in E. coli have a gain-of-function phenotype, whereas Szwedziak et al (2012) report that analogous mutants in B. subtilis FtsA suffer a loss of function. These results support the idea that FtsA self-association is related to its activity (Shiomi and Margolin, 2007), yet understanding how self-interaction regulates FtsA function clearly requires further study.The ability of eukaryotic cytoskeletal proteins to form long polymers is essential to their function, but the physiological relevance of long polymer formation by bacterial cytoskeletal proteins is now a topic of debate (Figure 1). For example, it has been hypothesized that FtsZ protofilaments wrap around the entire circumference of the cell to form the cytokinetic ring. However, recent studies using photoactivated localization microscopy (PALM) and electron cryotomography reveal a different model in which FtsZ forms a series of very short polymers that overlap to encompass the diameter of the cell (Li et al, 2007; Fu et al, 2010). MreB was also originally thought to form long-range helical polymers extending the length of the cell, but recent data obtained with more sophisticated microscopic techniques suggest that MreB is distributed in patches that move circumferentially and independently (White and Gober, 2012). It is not yet clear which of these models represents the true cellular architecture of MreB, although it is likely that some degree of MreB polymerization is still needed for function. It is notable that other bacterial homologs of actin and tubulin used for generating scaffolds or partitioning plasmid DNA, but not for essential cellular processes such as cell division and growth, tend to form long polymers that extend throughout the cell (Pogliano, 2008). The continued combined use of microscopic, biochemical, and genetic methods, as demonstrated by Szwedziak et al (2012) will enhance future understanding of ancestral tubulin and actin proteins in prokaryotes.Open in a separate windowFigure 1Bacterial actin and tubulin filaments involved in cell growth and division. (A) MreB (purple) has long been thought of as a spiral filament twisting along the cell length to control cell shape. Likewise, FtsZ protofilaments (blue) were once thought to wrap around the cell midpoint to organize the divisome. (B) Recent work using high-resolution microscopy has revealed that long cytoskeletal filaments are more likely to be short patches of polymers. The present work by Szwedziak et al (2012) has added FtsA actin-like filaments (green) to the model of possible divisome architecture.     

Flavin binding by bacterial luciferase: Affinity chromatography of bacterial luciferase

Immobilized FMN covalently attached to Sepharose-6B-hexanoate binds bacterial luciferase. Immobilized flavin is also effective in its reduced form as a substrate in the light emitting reaction catalyzed by luciferase.     

Protection of mice against bacterial infection by oral administration of bacterial lipopolysaccharide

The effect of orally administered bacterial lipopolysaccharide (LPS) on host resistance against bacterial infections was studied. LPS orally given for 5 consecutive days prior to infection caused no apparent toxic effect and protected mice against Pseudomonas aeruginosa and Listeria monocytogenes infections.     

Evolution of bacterial genomes

This review examines evolution of bacterial genomes with an emphasis on RNA based life, the transition to functional DNA and small evolving genomes (possibly plasmids) that led to larger, functional bacterial genomes.