健康妇女及3例细菌性阴道病患者阴道菌群的非培养分析

根据Amsel标准及Nugent标准,确诊筛选健康妇女及细菌性阴道病(bacterial vaginosis,BV)患者各3例.提取其阴道分泌物样本的总DNA,构建16S rRNA基因克隆文库,并对阳性克隆进行ARDRA和测序分析.结果表明,健康妇女样本的基因文库中,分别以卷曲乳酸杆菌(L.crispatus)和惰性乳酸杆菌(L. iners)的克隆子占较大比例,另外存在少量的阴道乳酸杆菌(L.vaginalis)或詹氏乳酸杆菌(L.jensenii)的克隆子.BV患者样本的基因文库中,克隆子所代表的菌种类型明显增多,但均以阴道加德纳氏菌(Gardnerella vaginalis)和阴道阿托波氏菌(Atopobium vaginae)的克隆子占较大比例,且无乳酸杆菌克隆子.说明健康妇女阴道菌群的种类单一,以乳酸杆菌占优势,L. iners为优势菌种之一;BV患者阴道菌群的种类复杂多样,但均以Gardnerella vaginalis及Atopobium vaginae共同占优势.     

细菌性阴道病患者阴道分泌物16SrDNA序列分析

目的分析健康妇女及细菌性阴道病(Bacterial vaginosis,BV)患者阴道分泌物16S rDNA序列。方法提取20例健康妇女及40例BV患者阴道分泌物标本中的总DNA,针对细菌16S rDNA保守区设计通用引物进行PCR扩增、克隆、测序,将获得的16S rDNA序列与美国国立生物技术信息中心(NCBI)数据库中的发表序列进行比对,分析克隆群中细菌种类和比例。结果通过阴道分泌物16S rDNA序列分析,发现健康妇女阴道分泌物中以卷曲乳酸杆菌(Lactobacillus crispatus),惰性乳酸杆菌(Lactobacillus iners),加氏乳酸杆菌(Lactobacillus gasseri)为优势菌种,而BV患者阴道菌群种类繁多,以加德纳菌属(Gardnerella)和奇异菌属(Atopobium vaginae)克隆子占较大比例,仅4例患者可见卷曲乳酸杆菌(Lactobacillus crispatus),其他患者均未见有乳酸杆菌克隆子且奇异菌属阴道病患者甲硝唑治疗疗效较差。结论健康妇女和BV患者阴道分泌物菌群种类有较大区别,BV患者在治疗前进行16S rDNA序列分析检测有较大的临床意义。     

阴道加德纳菌研究进展

细菌性阴道病(bacterial vaginosis,BV)主要由阴道加德纳氏菌(Gardnerella vaginalis,GV)与某些厌氧菌混合感染引起,伴有阴道分泌物性质改变的一组症候群,其病理特征无炎症病变和白细胞浸润。是广大妇女的常见病、多发病,占外阴阴道感染的30%~50%[1]。由于对BV病原菌认识的争议     

阴道加德纳菌检出率及唾液酸酶A基因携带与细菌性阴道病的关系

目的探究阴道加德纳菌(Gardnerella vaginalis)检出率及唾液酸酶A基因携带与细菌性阴道病(BV)的关系。方法选择2017年1月至2019年8月确诊的BV患者82例作为BV组,并随机选择同时期健康女性82例作为健康组,比较2组人群G. vaginalis检出率和唾液酸酶A基因携带情况,相关统计学资料分析其对BV发生的影响。结果BV组人群G. vaginalis阳性检出率高于健康组(χ²=11.511,P<0.05)。BV组人群共检出G. vaginalis 1、2、3、4、5、6和7型,其中2型占比最高;BV组人群G. vaginalis 2、3、4型占比率高于健康组(χ²=4.148,17.009,9.973,均P<0.05)。BV组人群唾液酸酶A基因携带率高于健康组(χ²=39.234,P<0.05)。较健康组人群,BV组PCR DGGE宽度更窄,肠道菌群条带数少(t=9.217,P<0.05)。结论G. vaginalis检出率和唾液酸酶A基因携带情况与BV发生相关,有待成为相关生物学治疗靶点。     

妊娠妇女与无性生活女性阴道分泌物 微生物组学分析

目的探讨妊娠妇女与无性生活女性阴道分泌物中菌群结构差异,为临床女性泌尿生殖系统疾病研究提供可靠的依据。方法运用高通量二代测序技术检测20例妊娠妇女和29例无性生活女性阴道分泌物中微生物;采用秩和检验进行组间显著性差异分析,使用生物信息学软件进行数据处理。结果 49份样本共注释出14个门、23个纲、33个目、28个科、38个属和23个种水平物种。妊娠妇女与无性生活女性阴道分泌物中菌群结构差异显著。妊娠妇女在种水平仅有Atopobium vaginae、Campylobacter ureolyticus、Lactobacillus coleohominis、德氏乳杆菌、瑞氏乳杆菌、惰性乳杆菌、罗伊乳杆菌、白假丝酵母菌和近平滑假丝酵母菌共9个物种。结论妊娠能引起女性阴道分泌物菌群结构发生改变,且菌种数量明显减少。Lactobacillus coleohominis和德氏乳杆菌可能在维持妊娠妇女阴道菌群平衡和自净作用中起到关键性作用。     

糖尿病患者阴道真菌菌群结构与生态分布研究

目的 探讨糖尿病(DM)患者阴道真菌菌群结构和生态分布特点.方法 采用真菌18S rDNA克隆文库法,对不同血糖控制情况下DM患者的阴道真菌18S rDNA序列进行克隆和测序,构建阴道真菌18S rDNA克隆文库,建立系统发育树,获取DM患者阴道真菌菌群的结构和组成特征.结果 DM患者和健康妇女阴道标本中存在3个真菌类群14个属共20个种系型的真菌菌群.健康妇女(H组)、HbA1c＜6.5％患者(DMa组)、HbA1c6.5％～7.5％患者(DMb组)和HbA1c＞7.5％患者(DMc组)的阴道真菌菌落分别存在7、9、13和16个OTUs.念珠菌属为各组研究对象阴道真菌菌群的优势菌属,其阳性克隆子在H组、DMa组、DMb组和DMc组的比例呈上升趋势.DM患者阴道白色念珠菌、光滑念珠菌和克柔念珠菌的比例不同程度升高,而阴道酿酒酵母菌和Uncultured fungi的比例显著降低(P＜0.05),伴随其他一些真菌菌种的出现和比例改变.结论 DM患者阴道真菌菌群分子生态结构发生不同程度改变,DM患者阴道真菌菌群紊乱程度与血糖控制情况关系密切.     

细菌性阴道病微生态与益生菌

细菌性阴道病(BV)是育龄妇女阴道炎的最常见原因,其特征是栖居在阴道内的乳酸杆菌减少导致阴道菌群平衡失调的复杂变化。用乳酸杆菌替代抗生素是治疗BV的一种有效的治疗措施。乳酸杆菌产生过氧化氢、乳酸、杆菌素能抑制引起BV的细菌生长。多胺在BV的病理机制中有重要作用。本研究拟综述BV患者阴道菌群的变化,免疫防御反应,阴道乳酸杆菌对BV的影响,多胺的意义,益生菌在治疗选择中的作用。     

阴道乳杆菌胶囊调整产后阴道菌群失衡的临床研究

目的研究产后6~8周妇女阴道微生物环境的变化特征及阴道菌群失衡的发病率,探讨应用阴道乳杆菌胶囊恢复产后妇女阴道内环境,预防生殖道感染发生的临床意义。方法收集1 053例产妇产后6~8周回访就诊时阴道分泌物,采用革兰染色法检查,进行Nugent评分,按Nugent评分系统对菌群分类,进一步诊断细菌性阴道病(BV),并与其孕早期产检时情况比对。结果 1 053例产后6~8周妇女Nugent评分正常521例,临界242例,有290例发生阴道炎症,细菌性阴道病205例,阴道假丝酵母菌74例和滴虫11例。疾病构成比以非特异性阴道炎为主,特异性阴道炎较为少见。所有妇女阴道p H均在5.0以上。在所有涂片中,被认为在维持正常阴道微生态平衡中起关键作用的乳杆菌数量和比例明显不足。产后BV的发病率(31.36%)高于孕早期产检时,产后妇女BV发病率较高,差异有统计学意义(P0.05)。予300例患者阴道内放置乳酸杆菌活菌胶囊,结果显示研究组阴道p H明显降低,接近正常女性阴道p H水平(P0.05),且阴道乳酸杆菌数量明显增多(P0.05);同时产后3个月、6个月阴道炎的发生率均明显低于对照组(P0.05)。结论产后妇女阴道乳酸杆菌量减少,菌群比例明显失调。阴道炎症以非特异性阴道炎为主,细菌性阴道病、真菌性阴道炎和滴虫性阴道炎较少见。乳杆菌胶囊作为生态治疗的活菌制剂,既能预防、治疗阴道感染,又无不良反应,还可以减少产后妇女阴道炎的复发率,值得临床推广应用。     

细菌性阴道病肠道菌群与阴道菌群16S rDNA测序分析

目的通过对细菌性阴道病(BV)患者肠道菌群及阴道菌群16S rDNA扩增子测序,分析其结构、多样性、相关性以及BV对肠道菌群的影响,为今后治疗BV提供新的思路。方法选取符合纳入标准的BV患者11例(BV组),健康者9例(C组),留存阴道分泌物及新鲜粪便进行16S rDNA基因检测分析。结果C组阴道菌群以乳杆菌属为主,BV与加德纳菌属、普雷沃菌属、Sneathia、窄食单胞菌属(Stenotrophomonas)、阿托波菌属、Shuttleworthia、巨型球菌属密切相关。BV组肠道、阴道菌群丰富度均高于C组。Alpha多样性分析中C组和BV组肠道菌群、阴道菌群的Shannon指数组间比较,χ²值为29.137, P=0.000<0.05,两组阴道菌群Shannon指数组间比较差异具有统计学意义(P<0.05),BV组高于C组。物种多样性曲线反映本研究样本测序数据量的合理性,表明BV组的肠道菌群多样性、丰富度均高于C组,主坐标分析表明C组肠道与阴道的菌群结构差距较大,BV组肠道与阴道的菌群结构有相似之处,且两组肠道菌群结构接近。BV组阴道菌群中厚壁菌门丰度较C组低,放线菌门、拟杆菌门较C组高;BV组肠道菌群中拟杆菌门丰度较C组低;C组肠道中拟杆菌门明显高于阴道,厚壁菌门明显低于阴道; BV组阴道菌群中放线菌门丰度高于C组,差异均具有统计学意义(P<0.05)。结论BV阴道菌群与肠道菌群具有相关性,BV可能引起肠道菌群结构比例和多样性的改变。     

HPV感染对患者阴道菌群及妊娠结局的影响

目的研究HPV感染对患者阴道菌群以及妊娠结局的影响。方法选择2017年9月至2018年9月在本院定期产检的孕妇,根据妊娠结局将孕妇分为正常妊娠组和不良妊娠组。收集两组患者一般资料及其阴道样本进行微生物检测及分析。结果不良妊娠组患者中HPV阳性患者比例明显高于正常妊娠组,正常妊娠组中HPV阴性和HPV阳性患者及不良妊娠组中HPV阴性患者阴道中乳杆菌(Lactobacillus)丰度最高,其次是加德纳菌(Gardnerella),普氏菌(Prevotella)和纤毛球菌(Sneathia)。不良妊娠组中HPV阳性患者阴道中乳杆菌(Lactobacillus)丰度最高,其次是加德纳菌(Gardnerella),芽孢杆菌(Bacillus),巨球菌(Megasphaera)和链球菌(Streptococcus)。不良妊娠组HPV阳性患者阴道中链球菌、巨球菌和芽孢杆菌丰度明显高于HPV阴性患者,乳杆菌和奇异菌(Atopobium)丰度明显低于HPV阴性患者。结论 HPV感染女性更易产生不良妊娠结局,其机制可能是HPV降低了阴道中乳杆菌丰度,增加阴道微生物群多样性和丰富度。     

Response of Gardnerella vaginalis biofilm to 5 days of moxifloxacin treatment

Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.     

Inerolysin, a cholesterol-dependent cytolysin produced by Lactobacillus iners

Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.     

Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

Bacterial vaginosis (BV), a common condition seen in premenopausal women, is associated with preterm labor, pelvic inflammatory disease, and delivery of low birth weight infants. Gardnerella vaginalis is the predominant bacterial species associated with BV, although its exact role in the pathology of BV is unknown. Using immunofluorescence, confocal and transmission electron microscopy, we found that VK2 vaginal epithelial cells take up G. vaginalis after exposure to the bacteria. Confocal microscopy also indicated the presence of internalized G. vaginalis within vaginal epithelial cells obtained from a subject with BV. Using VK2 cells and (35)S labeled bacteria in an invasion assay, we found that a 1 h uptake of G. vaginalis was 21.8-fold higher than heat-killed G. vaginalis, 84-fold compared to Lactobacillus acidophilus and 6.6-fold compared to Lactobacillus crispatus. Internalization was inhibited by pre-exposure of cells to cytochalasin-D. In addition, the cytoskeletal protein vimentin was upregulated in VK2 cells exposed to G. vaginalis, but there was no change in actin cytoskeletal polymerization/rearrangements or vimentin subcellular relocalization post exposure. Cytoskeletal protein modifications could represent a potential mechanism for G. vaginalis mediated internalization by vaginal epithelial cells. Finally, understanding vaginal bacteria/host interactions will allow us to better understand the underlying mechanisms of BV pathogenesis.     

Quantitative determination by real-time PCR of four vaginalLactobacillus species,Gardnerella vaginalisandAtopobium vaginaeindicates an inverse relationship betweenL. gasseriandL. iners

Background  

Most studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats forLactobacillus crispatus,L. gasseriandGardnerella vaginalisand developed new formats forAtopobium vaginae,L. inersandL. jenseniito obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years.     

Analysis of vaginal lactic acid producing bacteria in healthy women

Vaginal lactic acid-producing bacteria of 80 pre-menopausal women were studied by isolation on Blood and DeMan-Rogosa-Sharpe agar, PCR with group-specific primers for Lactobacillus-denaturing gradient gel electrophoresis (DGGE), and PCR with specific primers for V3 region in 16S rRNA-temporal temperature gel electrophoresis (TTGE). Conventional isolation method on media detected only one lactobacillus (Lactobacillus brevis) while TTGE detected only Lactobacillus sp. DGGE detected seven Lactobacillus species; L. coleohominis, L. crispatus, L. iners, L. reuteri, L. rhamnosus, L. vaginalis, and Leuconostoc lactis. L. acidophilus and L. gasseri, which are prevalent in Western women, were not detected in Korean women. Furthermore, L. rhamnosus, Leuc. lactis, L. coleohominis, and Weissella cibaria, which were not previously reported in the vaginal microbiota of Korean women, were detected. The five most prevalent LABs in vaginal microbiota in Korean women were L. iners, Enterococcus faecalis, L. crispatus, Leuc. lactis, and W. cibaria.     

Real-time PCR分析滴虫性阴道炎患者微生物组成的变化

目的探寻滴虫性阴道炎患者阴道微生物组成的特点,为临床诊治及减少疾病的复发提供参考依据。方法选取2016年5-6月复旦大学附属华东医院妇科门诊45岁以下确诊为滴虫性阴道炎的育龄期患者16例,以同期18例45岁以下育龄期健康女性为对照,采集患者和健康人上阴道壁1/3处的分泌物,进行常规临床分析和革兰染色,并以21种常见的阴道细菌的特异引物进行Real-time PCR检测,分析并比较这些细菌在滴虫性阴道炎患者和健康人阴道内的组成及变化。结果在18例健康人中,其中13例的阴道微生物组成以乳酸杆菌(Lactobacillus)为优势菌,3例的阴道微生物组成无明显优势菌群或者优势菌不为检测的21种常见阴道微生物,2例的阴道微生物组成以加德纳菌为优势菌;在16例滴虫性阴道炎患者中,只有2例患者的阴道微生物菌群以乳酸杆菌为优势菌,14例以厌氧菌如普雷沃属(Prevotella)、纤毛菌(Leptotrichia)和斯尼思菌(Sneathia)为优势菌。经Metastats进行组间差异分析,结果表明Lactobacillus crispatus、Leptotrichia amnionii、Eggerthella sp.和Peptostreptococcus sp.在两组之间差异有统计学意义(P≤0.05)。PCoA主成分析结果亦显示滴虫性阴道炎患者的阴道常见21种微生物组成与健康人有显著差别。结论滴虫性阴道炎患者阴道内微生物组成的多样性增加,普雷沃属、纤毛菌及斯尼思菌的明显增高可能滴虫性阴道炎的炎症发生有关,此结果对于临床治疗滴虫性阴道炎的及减少复发提供新的方向,具有一定的指导意义。