Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.     

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。     

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。     

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.     

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.     

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.     

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.     

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。     

Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences

AIMS: Lactobacilli are widely distributed in food and the environment, and some colonize the human body as commensal bacteria. The aim of this study was to determine the species of lactobacilli that colonize the vagina and compare them with those found in food and the environment. METHODS AND RESULTS: Thirty-five Lactobacillus strains from women from seven countries were isolated, and sequences from 16S rRNA genes were determined and compared with existing data in GenBank. A phylogenetic tree was achieved using the Neighbour-Joining method based on the analysis of 1465 nucleotides. The results showed that most vaginal isolates were L. crispatus, L. jensenii and L. gasseri. Some were L. vaginalis, L. fermentum, L. mucosae, L. paracasei and L. rhamnosus. Two isolates from a native American woman displayed distinct branches, indicating novel phylotypes. Few vaginal isolates matched food or environmental Lactobacillus species. CONCLUSIONS: Most women worldwide were colonized by three common Lactobacillus species: L. crispatus, L. jensenii and L. gasseri. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of vaginal Lactobacillus species richness and distribution in women worldwide may lead to the design of better probiotic products as bacterial replacement therapy.     

Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.     

Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut

AIMS: To evaluate the diversity of the Lactobacillus group in breast milk and the vagina of healthy women and understand their potential role in the infant gut colonization using the 16S rRNA gene approaches. METHODS AND RESULTS: Samples of breast milk, vaginal swabs and infant faeces were aseptically collected from five mothers whose neonates were born by vaginal delivery and another five that had their babies by caesarean section. After polymerase chain reaction (PCR) amplification using Lactobacillus group-specific primers, amplicons were analysed by denaturing gradient gel electrophoresis (DGGE). Clone libraries were constructed to describe the Lactobacillus group diversity. DGGE fingerprints were not related to the delivery method. None of the species detected in vaginal samples were found in breast milk-derived libraries and only few were detected in infant faeces. CONCLUSIONS: The bacterial composition of breast milk and infant faeces is not related to the delivery method. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been suggested that neonates acquire lactobacilli by oral contamination with vaginal strains during delivery; subsequently, newborns would transmit such bacteria to the breast during breastfeeding. However, our findings confirm, at the molecular level that in contrast to the maternal vagina, breast milk seems to constitute a good source of lactobacilli to the infant gut.     

Vaginal microbiome and epithelial gene array in post-menopausal women with moderate to severe dryness

After menopause, many women experience vaginal dryness and atrophy of tissue, often attributed to the loss of estrogen. An understudied aspect of vaginal health in women who experience dryness due to atrophy is the role of the resident microbes. It is known that the microbiota has an important role in healthy vaginal homeostasis, including maintaining the pH balance and excluding pathogens. The objectives of this study were twofold: first to identify the microbiome of post-menopausal women with and without vaginal dryness and symptoms of atrophy; and secondly to examine any differences in epithelial gene expression associated with atrophy. The vaginal microbiome of 32 post-menopausal women was profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Sixteen subjects were selected for follow-up sampling every two weeks for 10 weeks. In addition, 10 epithelial RNA samples (6 healthy and 4 experiencing vaginal dryness) were acquired for gene expression analysis by Affymetrix Human Gene array. The microbiota abundance profiles were relatively stable over 10 weeks compared to previously published data on premenopausal women. There was an inverse correlation between Lactobacillus ratio and dryness and an increased bacterial diversity in women experiencing moderate to severe vaginal dryness. In healthy participants, Lactobacillus iners and L. crispatus were generally the most abundant, countering the long-held view that lactobacilli are absent or depleted in menopause. Vaginal dryness and atrophy were associated with down-regulation of human genes involved in maintenance of epithelial structure and barrier function, while those associated with inflammation were up-regulated consistent with the adverse clinical presentation.     

Analysis of vaginal lactobacilli from healthy and infected Brazilian women

Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H(2)O(2)-producing lactobacilli were not associated with protection against VVC.     

Variability of bacterial biofilms of the "tina" wood vats used in the ragusano cheese-making process

Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.     

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus

AIMS: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. METHODS AND RESULTS: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8-357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. CONCLUSION: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.     

利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。     

Media and process parameters affecting the growth, strain ratios and specific acidifying activities of a mixed lactic starter containing aroma-producing and probiotic strains

AIMS: The effects of medium-composition and fermentation parameters on the properties of mixed mesophilic starters were studied. The starter was composed of Lactococcus lactis ssp. lactis (L. lactis), Lactococcus lactis ssp. cremoris (L. cremoris), Lactobacillus rhamnosus (Lact. rhamnosus) and Leuconostoc mesenteroides ssp. cremoris (Leuc. cremoris). METHODS AND RESULTS: The media used were reconstituted skim milk (RSM), and whey-based media with either citrate or phosphate buffers. The fermentation parameters were incubation temperature (22 degrees C or 32 degrees C), no pH control, and pH control in pH zones of either pH 6.0-5.8 or pH 6.0-5.2. The starter properties were strain ratio, specific acidifying activity (SAA), total population, residual carbohydrates and organic acids produced. The growth of L. lactis was favoured under pH control in whey-based media. High concentrations of Lact. rhamnosus were favoured in whey-based media prepared at 32 degrees C. The highest contents of Leuc. cremoris were obtained in starters prepared in RSM at 22 degrees C without pH control. Starters prepared under pH control gave the highest populations and made it possible for significantly lower inoculation rates (IR) to be used to carry out subsequent milk fermentations. However, the SAA of starters prepared under pH control were lower than the SAA of starters grown without any pH control. CONCLUSIONS: None of the conditions enabled the strain ratio at inoculation to be maintained. The data show that it is possible to prepare a mesophilic starter that has a significant probiotic Lact. rhamnosus content; this starter could be used in the preparation of probiotic-containing cheeses or in Leuc. cremoris for aroma production in fermented milks. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on what should be expected with respect to strain ratios and IR if cheesemakers decide to shift their aroma-producing starter production method from the traditional 'milk-based without pH control' method to whey-based media used with pH-zone control strategies.     

Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

Analysis of the presence ofprtR proteinase gene in natural isolates ofLactobacillus rhamnosus

The region of the prtR gene coding for the active site of PrtR proteinase was detected in natural isolates of lactobacilli, previously determined as Lactobacillus rhamnosus. This region was present in all L. rhamnosus strains with proteolytic activity. The PCR primers used were constructed on the basis of the sequence of the catalytic domain of the prtR proteinase gene. These primers generated in colony-PCR procedure specific 611 1-bp product with DNA from natural isolates of L. rhamnosus. No PCR amplifications using these primers were obtained for closely related bacteria of genus Lactobacillus, regardless of their proteolytic activity. In addition, these primers could be used singly or in multiplex PCR together with the Lactobacillus genus-specific primers. Compared with the other proteinases within the genus Lactobacillus (PrtP, PrtB and PrtH) which retained the activity in cell-free proteinase extracts, PrtR proteinase showed proteolytic activity only under in vivo conditions (whole cells of the producing strains).