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。     

阴道菌群多样性与宫颈癌的相关性研究

摘要 目的：研究宫颈癌疾病中女性阴道菌群的构成变化,探索具有预防宫颈癌发生的阴道益生菌。方法：选取新疆医科大学附属肿瘤医院和社区募集的24例健康女性和43例宫颈癌女性。采用16S rDNA和宏基因组测序技术分析其阴道菌群构成。结果：两组女性阴道菌群在chao1指数和ASV number中无统计学差异,提示群落丰度相似,在表示物种多样性的Shannon指数和Simpson指数中宫颈癌组显著增高,差异具有统计学意义。在微生物分类的门水平,两组阴道微生物均以厚壁菌门为主要物种,但宫颈癌组（45%）较健康组（91%）比例有所下降,其余如变形菌门和放线菌门等所占比例均显著升高。在种水平,宫颈癌组中的乳酸杆菌属（14.2%）较健康组（81%）比例下降明显,在宏基因组测序基础上,利用LEfSe及LDA判别分析得出在健康女性中卷曲乳杆菌具有益生菌特征意义,并可能在ABC转运体功能中发挥益生效应。结论：宫颈癌女性具有阴道微生物多样性增加,乳酸杆菌水平减少的特点,其中,通过宏基因组测序技术探究微生物种水平的差异物种得出卷曲乳杆菌在ABC转运体功能中具有潜在预防宫颈癌发生的益生菌作用,可为今后临床在调节阴道微生态角度预防宫颈癌发生提供理论依据。     

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.     

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。     

Characteristics of Vaginal Microbiome in Women with Pelvic Inflammatory Disease in Korea

Human vaginal microorganisms play an important role in maintaining good health throughout the human life cycle. An imbalance in the vaginal microbiota is associated with an increased risk of pelvic inflammatory disease (PID). This study aimed to characterize and compare vaginal microbial profiles of premenopausal Korean women with and without PID. 74 Korean premenopausal female vaginal samples were obtained; 33 were from healthy women (a control group) and 41 from PID patients. Vaginal fluid samples were collected from the vaginal wall and posterior cervix and then analyzed by 16S ribosomal ribonucleic acid (rRNA) gene-based amplicon sequencing. Results showed a significant difference between the vaginal microbial communities of the two groups (Jensen-Shannon, p = 0.014; Bray-Curtis, p = 0.009; Generalized UniFrac, p = 0.007; UniFrac, p = 0.008). Lactobacillus accounted for the highest percentage (61.0%) of the control group but was significantly decreased (34.9%) in PID patients; this was the most significant difference among all bacterial communities (p = 0.028, LDA effect size = 5.129). In addition, in the PID patient group, species diversity significantly increased (Simpson, p = 0.07) as the proportion of various pathogens increased evenly, resulting in a polymicrobial infection. Similarly, lactate, which constituted the highest percentage of the organic acids in the control group, was significantly decreased in the PID patient group (p = 0.04). The present study’s findings will help understand PID from the microbiome perspective and are expected to contribute to the development of more efficient PID diagnosis and treatment modalities.     

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。     

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。     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

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。     

Obstetric and Gynecological Diseases and Complications Resulting from Vaginal Dysbacteriosis

Accurate knowledge of the composition and ecology of vaginal microbial environment of a healthy woman is necessary for the understanding of normal flora and how to reduce the risk for diseases. Vagina and its microflora form a balanced ecosystem in which dominated bacteria are vaginal lactobacilli. There are dynamic changes in this ecosystem having structure and composition depending on many factors. The term dysbacteriosis defines any movement outside the normal range for the given biotope of obligate and/or facultative microflora. Such a change in the quantity and quality of the respective microbial balance is fraught with danger and requires correction and recovery. The purpose of this overview is to examine obstetric and gynecological diseases that can cause vaginal impaired microbial balance. Vaginal dysbacteriosis is a cause, predecessor, and often also consequence of vaginal infections. In essence, any vaginal infection can be seen as dysbacteriosis, developed to the most severe extent. Here, there is a dominant microorganism other than lactic acid bacteria in the vagina (clinically manifested or not, respectively), depletion of defense mechanisms of the vagina associated with the shift of lactobacilli from their dominant role in the vaginal balance, decrease in their number and species diversity, and a resulting change in the healthy status of the vagina. Vaginal dysbacteriosis can be found in pathogenetic mechanism, whereby many obstetric and gynecological diseases develop. Most of these diseases lead directly to increased maternal and infant morbidity and mortality, so it is important to understand the reasons for them and the arrangements for their prevention.     

Importance of associated microorganisms in the development of acute pneumonia

Sputum samples obtained from 106 patients with acute pneumonia have been studied by the quantitative microbiological method. Different microbial associations have been shown to play an important role in the development of acute pneumonia (67.9%). Microbiological studies have revealed the prevalence of pneumococci in the etiological structure of the disease. Staphylococci, hemophilic bacteria and Neisseria have been found to take part in the development of acute pneumonia in a lesser number of cases and more often occur in combination with pneumococci or form different associations themselves. The so-called "etiological" type of microbial associations has been determined, two or three microbial species being isolated at high concentrations (10(6) and over) and the percentage of patients with such combinations being 59.7 +/- 5.8%. The analysis of the clinical course of the disease has revealed that the presence of different microbial species in patients linked, to a great extent, with the clinical picture of the disease. These data may be used for prescribing adequate etiotropic therapy and for prognostication. Quantitative bacteriological studies carried out in the dynamics of the disease permit timely detection of changes in the microflora of the respiratory tracts, the evaluation of the effectiveness of antibacterial therapy in the process of its implementation and the rational correction of therapeutic measures.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

定君生联合甲硝唑治疗滴虫性阴道病的临床疗效观察

目的 通过定君生联合甲硝唑以及单独用药治疗滴虫性阴道病的临床疗效观察,探讨联合用药对滴虫性阴道病的疗效.方法 选择诊断明确的滴虫性阴道病患者150例,随机分为3组,试验组采用甲硝唑联合定君生的治疗,对照组Ⅰ采用定君生单独用药,对照组Ⅱ采用甲硝唑治疗,观察治疗后的症状、体征、滴虫和阴道的pH的变化.结果 3组患者有效率差异有统计学意义(P＜0.05),起效时间和复发率差异有统计学意义(P＜0.01).结论 定君生联合甲硝唑治疗滴虫性阴道病,疗效好,有利于快速恢复阴道的微生物环境的平衡,降低复发率.     

Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation.

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.     

Methods for quantitative and qualitative evaluation of vaginal microflora during menstruation

The quantitative and qualitative changes in the bacterial flora of the vagina during menstruation have received inadequate study. Similarly, the effect of vaginal tampons on the microbial flora as well as the relationship between the microbial flora of the vagina and that of the tampon has not been adequately evaluated. The purposes of the present study were (i) to develop quantitative methods for studying the vaginal flora and the flora of tampons obtained during menstruation and (ii) to determine whether there were differences between the microflora of the tampon and that of the vaginal vault. Tampon and swab samples were obtained at various times from eight young healthy volunteers for 8 to 10 menstrual cycles. Samples consisted of swabs from women wearing menstrual pads compared with swab and tampon samples taken at various times during the menstrual cycle. Samples were analyzed for total facultative and anaerobic bacterial counts, and the six dominant bacterial species in each culture were identified. Statistical evaluation of the results indicates that total bacterial counts decreased during menstruation and that swab and tampon samples yielded similar total counts per unit weight of sample. The numbers of bacteria in tampons tended to be lower than in swabs taken at the same time. Overall, during menstruation, the concentrations of lactobacilli declined, but otherwise there was little difference among the species found during menstruation compared with those found in intermenstrual samples. Cotton tampons had little discernible effect on the microbial flora.     

Specific features of normal vaginal microflora in girls of preschool age

The study of the vaginal microbial cenosis in 20 healthy girls aged 3-7 years did not confirm the notion on the dominating role of cocci (including epidermal staphylococci). The associations of 2-5 different microorganisms represented by more than 20 species in an amount of 4-6 Ig PFC/g of discharge were established. In the overwhelming majority of the examinees (84.2%) the microbial associations of the vagina were found to contain bifidobacteria. Gram positive cocci (staphylococci and streptococci) took the 2nd and 3rd places in the isolation rates and were detected in vaginal associations in 78.9% of the girls. Staphylococci were represented by 5 coagulase-negative staphylococcal species with S. simulans and S. epidermidis prevailing. Hemolytic streptococci variants alpha and beta were isolated in the proportion of 2:1. The latter belonged to serogroups C and F. No S. aureus, Lactobacillus sp., streptococci of groups A and B, yeast-like fungi were detected. Genital mycoplasms (M. hominis) could rarely be found in the vaginal discharge of the girls aged 3-7 years (5.3%). No resident and transitory components could be isolated from the normal vaginal microflora and no quantitative domination of any bacterial species (genus) was shown. The concentrations of all organisms in this association were moderate or low.     

Black-pigmented Gram-negative anaerobes in genito-urinary tract and pelvic infections

Abstract Black-pigmented Gram-negative anaerobes are part of the normal vaginal flora and contribute to a range of superficial and deep genital infections. Prevotella melaninogenica is found in moderate numbers (10⁴⁻⁶ cfu/g) in healthy women; the numbers and detection rates increase in anaerobic vaginosis (where it may be a significant contributor to changed microbial metabolic activity that gives the signs and symptoms of this condition) and in other vaginal infective conditions. Pr. melaninogenica is also part of the mixed flora in deep pelvic infections: endometritis, post-partum and post-abortal uterine infections; salpingitis and tubo-ovarian abscesses; PID and pelvic abscesses. Porphyromonas asaccharolytica is probably not a vaginal commensal, but may be isolated from patients with vaginal or pelvic disease. It is more specifically associated with superficial abscesses (e.g. Bartholin's abscess) and ulcers of the genitalia and perineum. P. asaccharolytica was the commonest species isolated from men with genital ulcers of various primary causes and ranging in severity from superficial balanitis/balanoposthitis to synergic gangrene.     

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development

Background  We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates.
Methods  Vaginal and rectal microflora in Chinese rhesus macaques ( Macaca mulatta ) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization.
Results  Chinese rhesus macaques harbored abundant vaginal Lactobacillus , with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals.
Conclusions  The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus -based topical microbicides.     

阴道微生态宏基因组学研究进展

女性阴道内寄居着多种微生物群落,这些微生物种群间的平衡状态与妇女阴道疾病的发生密切相关。鉴定女性阴道菌群结构多样性的特征,有助于了解其在阴道疾病发生和转归中所发挥的作用。目前基于16SrRNA的聚合酶链式反应(PCR)及宏基因组相关技术在微生物群落研究中被广泛运用,这不仅可以帮助人们最大程度地获得阴道菌群的宏基因组信息,还可有效弥补单纯微生物培养法所产生的实验数据不充足等弊端。本文对阴道微生物菌群多样性的研究中应用的宏基因组学技术如基因测序、变性/温度梯度凝胶电泳(DGGE/TGGE)、分子克隆、末端限制性酶切长度多态性(T-RFLP)等进行综述。     

阴道炎患者乳酸杆菌数量变化与临床的研究

目的探讨阴道炎患者的阴道乳酸杆菌数量的变化,以指导临床治疗。方法用LBS培养基分离乳酸杆菌,观察阴道炎患者乳酸杆菌变化的情况。结果172例阴道炎病例中,乳酸杆菌数量平均数明显减少。结论乳酸杆菌数量的减少、阴道菌群的改变是阴道炎发病的重要原因,重新构建阴道正常菌群是阴道炎治疗的重点。