利用X-Neu5Ac为底物测定唾液酸酶活性在诊断细菌性阴道病的价值

目的利用5溴-4氯-3吲哚乙酰基神经氨酸盐（X-Neu5Ac）为底物测定阴道唾液酸酶活性诊断细菌性阴道病（bacterial vaginosis,BV）的价值.方法健康妇女30例,临床Amsel法诊断为BV的患者45例,共计75例进行了阴道分泌物分析和检测,并与唾液酸酶活性法诊断作了对比研究.取阴道分泌物作为标本分别进行唾液酸酶活性和阴道菌群定量分析,检测细菌包括乳酸杆菌、类杆菌、肠杆菌、葡萄球菌、肠球菌和阴道加德纳菌.唾液酸酶活性测定利用的底物为X-Neu5Ac,特异活性用其产物 ——甲氧基苯酚的纳摩尔数来表示.结果阴道液唾液酸酶活性测定诊断细菌性阴道病的敏感性、特异性、阳性预期值和阴性预期值分别为88.9%、90%、93%和84.3%.唾液酸酶法在检测细菌性阴道病上和传统的Amsel法比较,差异无显著性（P＞0.05）.唾液酸酶阳性组Gv活菌数（6.96 log CFU/g）明显高于唾液酸酶阴性组（2.05 log CFU/g）（P＜0.01）.唾液酸酶阳性组产H2O2阴道乳杆菌（LB＋）活菌数（4.26 Log CFU/g）明显低于唾液酸酶阴性组（8.66 Log CFU/g）（P＜0.01）.唾液酸酶阳性组与唾液酸酶阴性组两组的阴道液中需氧菌活菌数差异无显著性（P＞0.05）,主要包括金黄色葡萄球菌、肠球菌和肠杆菌.结论利用X-Neu5Ac作为唾液酸酶的底物测定唾液酸酶活性的方法是诊断细菌性阴道病的有效检测方法.阴道内唾液酸酶活性增强,厌氧菌数量增加,LB＋数量减少,提示BV发生恶化.     

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。     

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。     

治疗前后加德纳菌载量 对细菌性阴道病患者复发风险的预测价值

目的评价治疗前后加德纳菌载量预测细菌性阴道病(bacterial vaginosis,BV)患者复发风险的临床价值。方法将2017年1月至2018年1月于我院就诊的312例BV患者纳入研究,根据治疗后BV是否复发分为复发组(114例)及未复发组(198例),分别比较两组患者治疗前后阴道分泌物的pH值、Nugent评分、乳杆菌数量和加德纳菌载量,并应用受试者工作曲线(ROC)对上述指标预测BV复发的诊断价值及诊断效能进行评价。结果治疗前及治疗后复发组患者的pH值(t=2.187、3.822,均P0.05)、Nugent评分(t=1.976、5.611,均P0.05)、加德纳菌载量(t=5.089、16.204,均P0.05)均明显高于未复发组,乳杆菌数量(t=8.245、8.899;均P0.05)明显低于未复发组;多因素Logistic回归分析显示治疗后阴道分泌物Nugent评分及加德纳菌载量均是BV复发的独立危险因素,而乳杆菌数量为保护因素;对上述指标预测BV复发的相关性进行分析,ROC曲线显示,加德纳菌载量预测BV复发的AUC为0.892,明显高于pH值、Nugent评分、乳杆菌数量(AUC=0.580、0.648、0.772),诊断最佳截点为1.761×10~5 copy/mL,此时的敏感性和特异性分别为93.5%和75.4%。结论 BV患者治疗后的加德纳菌载量在预测其复发方面具有较高的敏感性及特异性,可用于预测BV复发。     

需氧菌阴道炎、细菌性阴道病阴道菌群分析

用阴道分泌物直接涂片、革兰染色、选择性培养分离和细菌预成酶谱分析等微生物学方法和生物化学方法,分析了正常女性、AV、BV患者的阴道菌群。正常女性阴道中以乳酸杆菌为主,产H2O2乳酸杆菌的检出率89%,AV的致病菌主要是革兰阳性率需氧菌、检出率可达80%,其中金黄色葡萄球菌,粪肠球菌和埃希氏大肠菌较为常见,B族链球菌的检出率较低;BV的致病菌主要是厌氧菌,以革兰阴性厌氧菌的检出率最高,加德纳菌的检出率不足40%。48例BV患者中有8例合并AV感染,31例AV患者中有6例合并BV感染。细菌谱分析发现,AV患者易感染消化链球菌、普雷沃菌等厌氧菌和加德纳菌,BV患者易感染粪肠球菌、大肠埃希菌等需氧菌。     

乳杆菌活菌胶囊联合氨苄西林对产褥期阴道淋球菌感染患者的治疗

目的观察并评价乳杆菌活菌胶囊联合氨苄西林治疗产褥期阴道淋球菌感染患者的效果。方法将81例产褥期阴道淋球菌感染患者以随机数表分为常规组(40例)和联合组(41例)。常规组患者给予氨苄西林治疗,联合组患者给予乳杆菌活菌胶囊联合氨苄西林治疗。对比两组患者症状缓解时间,治疗前后阴道灌洗液肿瘤坏死因子α(TNFα)和白介素6(IL6)水平及阴道pH,同时观察治疗后阴道内乳杆菌检出情况,患者疗效和随访12个月内的复发率。结果联合组患者下腹疼痛、阴道分泌物增多、阴道口红肿、阴道口疼痛、显脓性白带症状缓解时间均短于常规组(均P<0.05)。2组患者治疗后阴道灌洗液TNFα、IL6水平和阴道pH均显著降低,且联合组以上指标水平均低于常规组[(42.45±10.20)ng/L vs(107.66±12.59)ng/L、(33.30±6.71)ng/L vs(59.74±7.99)ng/L、(4.27±0.25) vs(5.47±0.29)](均P<0.05)。2组患者治疗后阴道内乳杆菌检出情况及患者疗效差异均有统计学意义(均P<0.05),且联合组患者阴道内乳杆菌3+/4+者构成比明显高于常规组[82.93% vs 62.50%](P<0.05)。联合组患者总有效率明显高于常规组(97.56% vs 82.50%),而其复发率低于常规组(0.00% vs 15.15%)(均P<0.05)。结论乳杆菌活菌胶囊联合氨苄西林能够加快产褥期阴道淋球菌感染患者症状缓解,控制炎症反应,降低阴道pH;同时改善患者阴道内乳杆菌定植情况,疗效理想,对患者复发率也有积极的控制作用。     

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     

492例细菌性阴道病患者加德纳菌检测与药敏分析

目的了解当地女性细菌性阴道病(BV)患者中阴道加德纳菌(GV)的感染情况以及GV对常用抗菌药物的敏感性,为BV的诊断和治疗提供依据。方法对收集的492例BV患者和151例正常健康妇女的阴道分泌物进行GV的分离培养与鉴定,并对分离菌进行常用抗菌药物敏感性试验。结果 492例BV患者GV阳性率为27.8%,健康妇女GV阳性率为7.9%,两者比较差异有统计学意义(P<0.05)。分离的GV对万古霉素和克林霉素均较敏感,对头孢噻肟、四环素、左氧氟沙星、氨苄青霉素、红霉素、甲硝唑的敏感性依次降低。结论 GV为当地BV患者的重要病原菌之一,且对万古霉素和克林霉素均较敏感,对甲硝唑的敏感性最低。     

乳杆菌泡腾片治疗细菌性阴道病

目的观察10例BV患者采用了信谊制药总厂生产的阴道嗜酸乳杆菌泡腾片1个疗程(7 d)后阴道菌群的变化。方法采用7种选择或非选择培养基对阴道分泌物的细菌总数、乳杆菌等7个指标进行检测。结果治疗后阴道中乳杆菌显著增多,厌氧革兰阴性杆菌显著减少(两者P<0.001)。细菌总数、肠杆菌、酵母菌和阴道加德纳菌均减少(P<0.01)。结论信谊阴道嗜酸乳杆菌泡腾片具有较好的调整阴道微生态平衡的作用。     

乳杆菌属细菌对宫颈上皮内瘤变患者阴道菌群的影响

目的分析乳杆菌属细菌对宫颈上皮内瘤变(CIN)患者阴道菌群的影响,为该类患者的治疗提供参考。方法选择2019年1月至2020年1月我院收治的CIN患者137例,根据组织学病理结果分为CIN Ⅰ组(84例)和CIN Ⅱ、Ⅲ组(53例)。选择同期100例宫颈检查正常者为对照组。比较各组对象阴道菌群数量。根据患者阴道内乳杆菌数量将患者分为高数量组和低数量组,比较2组患者阴道菌群数量。结果CIN Ⅰ组患者阴道厚壁菌门数量低于CIN Ⅱ、Ⅲ组和对照组,而放线菌门数量高于CIN Ⅱ、Ⅲ组和对照组(均P<0.05)。CIN Ⅱ、Ⅲ组患者阴道乳杆菌属数量高于CIN Ⅰ组和对照组(均P<0.05)。CIN Ⅰ组患者阴道加德纳菌属、奇异菌属数量高于CIN Ⅱ、Ⅲ组和对照组(均P<0.05)。对照组对象阴道菌群Chao、ACE指数均低于CIN Ⅰ组和CIN Ⅱ、Ⅲ组(均P<0.05),同时CIN Ⅱ、Ⅲ组患者阴道菌群Chao、ACE指数均低于CIN Ⅰ组(均P<0.05)。高数量组患者阴道加德纳菌属、奇异菌属占比均低于低数量组(均P<0.05)。结论阴道中乳杆菌属数量的改变易影响阴道菌群,使其抗感染能力下降,增加女性CIN的风险。     

Binding of catalase by Gardnerella vaginalis

Previous work has demonstrated that Gardnerella vaginalis can utilize catalase as a sole source of iron. In this study, the interaction between G. vaginalis cells and catalase was investigated. G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled catalase using a solid phase dot blot assay. An increase in catalase binding was observed from cells grown under iron-restrictive conditions. Western blot analysis of G. vaginalis proteins resulted in the detection of a putative catalase-binding protein with an estimated molecular mass of 128 kDa. The 128-kDa catalase-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis suggesting this protein may be surface-exposed.     

Screening of Compounds against Gardnerella vaginalis Biofilms

Bacterial vaginosis (BV) is a common infection in reproductive age woman and is characterized by dysbiosis of the healthy vaginal flora which is dominated by Lactobacilli, followed by growth of bacteria like Gardnerella vaginalis. The ability of G. vaginalis to form biofilms contributes to the high rates of recurrence that are typical for BV and which unfortunately make repeated antibiotic therapy inevitable. Here we developed a biofilm model for G. vaginalis and screened a large spectrum of compounds for their ability to prevent biofilm formation and to resolve an existing G. vaginalis biofilm. The antibiotics metronidazole and tobramycin were highly effective in preventing biofilm formation, but had no effect on an established biofilm. The application of the amphoteric tenside sodium cocoamphoacetate (SCAA) led to disintegration of existing biofilms, reducing biomass by 51% and viability by 61% and it was able to increase the effect of metronidazole by 40% (biomass) and 61% (viability). Our data show that attacking the biofilm and the bacterial cells by the combination of an amphoteric tenside with the antibiotic metronidazole might be a useful strategy against BV.     

Prevalence of Gardnerella vaginalis: an estimate

To assess the prevalence of Gardnerella vaginalis in the community 300 women aged 16-59 were randomly selected from a general practice''s age-sex register and invited to attend for a health check. Out of 282 women who were eligible to attend, 192 did so. They were asked whether they had any vaginal symptoms, and swabs were taken from 182 women for culture for G vaginalis. Sixty women were positive for G vaginalis, of whom 26 had symptoms.Infections with G vaginalis may be present in women who have no symptoms. By careful questioning, examination, and side room testing general practitioners may be able to diagnose these infections in such women consulting them for other reasons.     

Identification of a Gardnerella vaginalis Hemoglobin-Binding Protein

Previous studies have shown that Gardnerella vaginalis can utilize human hemoglobin as a sole source of iron. In this study, the interaction between human hemoglobin and G. vaginalis cells was investigated. With a solid phase dot blot assay, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human hemoglobin. A human hemoglobin-binding protein with an estimated molecular weight of 124 kilodaltons (kDa) was detected by Western blot analysis of G. vaginalis proteins. The hemoglobin-binding activity of this protein was found to be heat stable and was observed in G. vaginalis cells grown under iron-restrictive and iron-replete conditions. The 124-kDa hemoglobin-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis, suggesting that this protein was surface exposed. Received: 26 June 2000 / Accepted: 21 July 2000     

Haemagglutination and tissue culture adhesion of Gardnerella vaginalis

Six strains of Gardnerella vaginalis were studied to examine the adhesin-receptor mechanism involved in their attachment to human red blood cells and an epithelial tissue culture cell line (McCoy). The adhesins involved in the attachment of the bacteria to each of these cells were proteinaceous but showed marked differences after various chemical or physical treatments, indicating that separate adhesins were present. Haemagglutinating strains were more hydrophobic than tissue-culture-adherent strains. Haemagglutination of human red blood cells by strains of G. vaginalis was inhibited by galactose, lactose, N-acetylneuraminic acid and phosphatidylserine. In contrast, the tissue-culture adherence of strains was not inhibited by these substances.     

Pore-forming and haemolytic properties of the Gardnerella vaginalis cytoiysin

The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 × 10⁶ HU mg^?1 being obtained. The toxin-induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose-response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol^?1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh-haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens theta-toxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram-positive and Gram-negative bacteria, respectively. The toxin shares several features with the family of the so-called ‘sulphydryl-activated’ cytolysins produced by Gram-positive bacteria, although Gvh does not truly belong to this family, being deactivated by β-mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.     

Detection of Gardnerella vaginalis on vaginal smears by immunofluorescence

An indirect fluorescence antibody (IFA) test was developed for the detection of Gardnerella vaginalis. Antisera were prepared in rabbits by using five strains of G. vaginalis. A pool of the antisera was tested for specificity with a variety of isolates known to colonize the human vagina and (or) morphologically resemble G. vaginalis. Six heterologous bacterial isolates reacted with the pooled antiserum at dilutions of 1:10, but none reacted at the working dilution of 1:200. Vaginal swab specimens were collected from symptomatic and asymptomatic patients in order to further evaluate the IFA procedure. The presence of G. vaginalis in the specimens was determined both by culture and by the IFA procedure. Absorbed antisera reacted with all isolates of G. vaginalis tested. In a clinical trial the IFA procedure detected the presence of G. vaginalis in smears from 23 (24.2%) of the patients with nonspecific vaginitis (NSV), from 22 (29.8%) of the asymptomatic individuals tested, and from 3 patients with vaginitis other than NSV. The presence of G. vaginalis in smears as detected by the IFA procedure was confirmed by cultures in all cases using Vaginalis agar supplemented with colistin and nalidixic acid (V-CNA). It is suggested that the IFA procedure may be of use in conjunction with V-CNA in epidemiological studies of the carriage and transmission of G. vaginalis in human populations. It appears that the IFA procedure, at least in our hands, is a useful test for the rapid detection of G. vaginalis even when this microorganism is not the predominant colonizer of the human vagina.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

Abstract The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al, 1991) FEBS Lett. 283, 317–320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 Å and 28 Å in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K⁺ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca²⁺ concentration, suggesting a Ca²⁺ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca²⁺ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.