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。     

阴道微生物组：种群特征与疾病干预治疗

高通量测序技术的发展显著加快了对人类微生物组的理解。将人体微生物组与疾病关联,力求阐明疾病的发生进程,是推进个性化精准医疗的重要研究方向。近年来,栖居于女性阴道的微生物菌群日益受到关注,发现其生态失调与疾病发生、演变密不可分。文中综述了阴道微生物组与生殖道疾病发生、进展和治疗的最新进展,同时对阴道微生物组培养组、益生菌工程化改造以及合成菌群在阴道微生物组学研究以及疾病干预与治疗方面的前景进行了展望。     

Vaginal microbial diversity among postmenopausal women with and without hormone replacement therapy

Urogenital infections in postmenopausal women remain problematic. The use of estrogen replacement therapy has been shown to lower these infection rates, corresponding to increasing colonization by Lactobacillus species. Despite the gut's 500 microbial species and the proximity of the anus to the vagina, only a relatively few microbial strains appear to be able to colonize the urogenital area. In the present study, the sparsity of microbes in the vagina was confirmed by denaturing gradient gel electrophoresis analysis of swabs taken at time zero and monthly for 3 months from 40 postmenopausal subjects receiving Premarin (conjugated equine estrogen in combination with progesterone) hormone replacement therapy (HRT) and 20 who were not on HRT. Lactobacilli were recovered from the vagina of 95% or more women in both groups, but in the HRT group, Lactobacillus were more often the dominant and only colonizers and significantly fewer bacteria with pathogenic potential were found. The incidence of bacterial vaginosis was significantly lower in the HRT group than in the non-HRT-treated women (5.6% versus 31%). The use of HRTs has recently come under criticism. The ability of drugs such as Premarin to help recover the lactobacilli vaginal microbiota appears to be at least one benefit of HRT use. In women not using HRTs, use of probiotics may be the only way to restore a nonpathogen-dominated flora.     

阴道菌群移植治疗细菌性阴道炎的发展现状及展望

细菌性阴道炎 (Bacterial vaginosis,BV) 是由患者阴道内菌群失调导致的一类疾病。当前常规的抗生素疗法可进一步加剧阴道菌群失衡、破坏阴道酸性环境、导致耐药,因此其对BV治愈率低,复发率高。作为一种新兴的活菌疗法,阴道菌群移植 (Vaginal microbiota transplantation,VMT) 直接将健康妇女完整的阴道菌群“嫁接”给患者,可迅速恢复患者阴道菌群的平衡,改善患者的整体健康。文中对VMT的发展历程进行了回顾,讨论了VMT发展过程中面临的难题及发展方向,以期寻求新的治疗BV的策略,加速VMT的临床应用。     

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.     

Identification and plasmid profiles of Lactobacillus species from the vagina of 100 healthy women

Abstract It was hypothesized that Lactobacillus acidophilus might not be the dominant aerobic or microaerophilic Lactobacillus species in the human vagina, and that the dominant isolates are likely to express a variety of properties associated with colonization. To examine the question, vaginal swabs were collected from 100 healthy premenopausal women and cultured to detect the dominant aerobic or microaerophilic isolates of Lactobacillus . Only eight species were detected, with Lactobacillus jensenii being the dominant colonizers in the highest number (35) of women. Twelve different plasmid profiles were found amongst the isolates, but there was no association between plasmids and vaginal colonization or hydrogen peroxide production. Of the strains tested, few were resistant to the spermicidal agent nonoxynol-9. The data identify species within which strains may possess properties associated with maintenance of a healthy vaginal ecology     

Molecular methods to describe the spectrum and dynamics of the vaginal microbiota

The human vagina hosts a collection of microbes that is distinct from other human surfaces and mucosal sites, with reduced microbial diversity that is likely driven by the acidic environment. The microbial ecosystem of the vagina is dominated by lactobacilli in women without bacterial vaginosis (BV), and is characterize by increased species richness, diversity, and evenness in women with BV. The use of molecular, cultivation-independent methods to describe the bacterial biota of the human vagina has revealed many novel putative anaerobes in women with BV, and has demonstrated the almost ubiquitous nature of Lactobacillus iners which is found in most women regardless of BV status. A variety of molecular tools are being employed to study the vaginal microbiota, and each approach has distinct advantages and disadvantages that are reviewed. Longitudinal studies have demonstrated that the vaginal microbiota can be highly dynamic, with dramatic shifts in bacterial composition and concentrations in response to numerous endogenous and exogenous factors.     

The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.     

Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized,placebo-controlled trial in 64 healthy women

Urogenital infections afflict an estimated one billion people each year. The size of this problem and the increased prevalence of multi-drug resistant pathogens make it imperative that alternative remedies be found. A randomized, placebo-controlled trial of 64 healthy women given daily oral capsules of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 for 60 days showed no adverse effects. Microscopy analysis showed restoration from asymptomatic bacterial vaginosis microflora to a normal lactobacilli colonized microflora in 37% women during lactobacilli treatment compared to 13% on placebo (P=0.02). Lactobacilli were detected in more women in the lactobacilli-treated group than in the placebo group at 28 day (P=0.08) and 60 day (P=0.05) test points. Culture findings confirmed a significant increase in vaginal lactobacilli at day 28 and 60, a significant depletion in yeast at day 28 and a significant reduction in coliforms at day 28, 60 and 90 for lactobacilli-treated subjects versus controls. The combination of probiotic L. rhamnosus GR-1 and L. fermentum RC-14 is not only safe for daily use in healthy women, but it can reduce colonization of the vagina by potential pathogenic bacteria and yeast.     

Use of a vaginal probiotic suppository and antibiotics to influence the composition of the endometrial microbiota

The aim of the present study was to determine the status of the endometrial microbiota in patients with repeated implantation failure (RIF) and to assess treatment strategies for nondominant Lactobacillus (NLD) cases. A total of 392 patients with RIF were enrolled in this prospective cohort study (UMIN-CTR 000038582) and underwent endometrial microbiota analysis. Patients diagnosed with NLD were treated with a combination of oral and vaginal probiotics or oral prebiotics and antibiotics. The outcome was evaluated through re-analysis of the endometrial microbiota following treatment, and the results are presented as cure rates. NLD represented 44.9 % of the total endometrial microbiota in patients with RIF. The most commonly detected bacterium was Gardnerella vaginalis. The cure rates in the oral probiotics + oral prebiotics, antibiotics, oral probiotics + oral prebiotics + antibiotics, vaginal probiotic suppository, and vaginal probiotic suppository + antibiotics groups were 29.5, 33.33, 33.33, 43.6, and 78.6 %, respectively. Significant improvements were noted in the vaginal probiotic suppository + antibiotics group. Moreover, we revealed that approximately half of patients with RIF had NLD. Thus, the combination of a vaginal probiotic suppository and antibiotics may represent an effective treatment for NLD cases.     

Probiotics L. acidophilus and B. clausii Modulate Gut Microbiota in Th1- and Th2-Biased Mice to Ameliorate Salmonella Typhimurium-Induced Diarrhea

Gut microbiota play important role in maintaining health. Probiotics are believed to augment it further. We aimed at comparing effects of probiotics, Lactobacillus acidophilus (LA) and Bacillus clausii (BC) (a) on the gut microbiota abundance and diversity and (b) their contributions to control intestinal dysbiosis and inflammation in Th1- and Th2-biased mice following Salmonella infection. We report how could gut microbiota and the differential immune bias (Th1 or Th2) of the host regulate host responses when challenged with Salmonella typhimurium in the presence and absence of either of the probiotics. LA was found to be effective in ameliorating the microbial dysbiosis and inflammation caused by Salmonella infection, in Th1 (C57BL/6) and Th2 (BALB/c)-biased mouse. BC was able to ameliorate Salmonella-induced dysbiosis and inflammation in Th2 but not in Th1-biased mouse. These results may support probiotics LA as a treatment option in the case of Salmonella infection.

     

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.     

Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract

AIMS: The study of two human strains of Lactobacillus to be used as probiotics in the gastrointestinal tract. METHODS AND RESULTS: The Lactobacillus acidophilus UO 001 and Lact. gasseri UO 002, were resistant to the gastrointestinal conditions (pH 2 and 3, presence of pepsin, pancreatin or bile salts), the resistance was enhanced in the presence of skimmed milk. Additionally, adhered to Caco-2 cells through glycoproteins in Lact. gasseri and carbohydrates in the case of Lact. acidophilus. These strains are able to inhibit the growth of certain enteropathogens: Salmonella, Listeria and Campylobacter without interfering with the normal microbiota of the gastrointestinal tract, as stated by using the mixed culture and the spot agar test. Finally, strongly adherent Lact. gasseri were found to inhibit the attachment of Escherichia coli O111 to intestinal Caco-2 cells under the condition of exclusion. CONCLUSIONS: These results indicate that the two strains of Lactobacillus from human origin present important properties for survival in, and colonization of, the gastrointestinal tract, that give them potential probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Two strains of Lactobacillus isolated from human vagina of healthy premenopausal women could be promising candidates to be used in the preparation of probiotic products and for their use as health-promoting bacteria.     

Biphasic assembly of the murine intestinal microbiota during early development

The birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site-specific microbial communities post-natally. We characterized the distal gut microbiota from birth to weaning in mice. One-day-old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of Lactobacillus. By weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. Our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from Streptococcus to Lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. Mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development.     

Colonic microbiome is altered in alcoholism

Several studies indicate the importance of colonic microbiota in metabolic and inflammatory disorders and importance of diet on microbiota composition. The effects of alcohol, one of the prominent components of diet, on colonic bacterial composition is largely unknown. Mounting evidence suggests that gut-derived bacterial endotoxins are cofactors for alcohol-induced tissue injury and organ failure like alcoholic liver disease (ALD) that only occur in a subset of alcoholics. We hypothesized that chronic alcohol consumption results in alterations of the gut microbiome in a subgroup of alcoholics, and this may be responsible for the observed inflammatory state and endotoxemia in alcoholics. Thus we interrogated the mucosa-associated colonic microbiome in 48 alcoholics with and without ALD as well as 18 healthy subjects. Colonic biopsy samples from subjects were analyzed for microbiota composition using length heterogeneity PCR fingerprinting and multitag pyrosequencing. A subgroup of alcoholics have an altered colonic microbiome (dysbiosis). The alcoholics with dysbiosis had lower median abundances of Bacteroidetes and higher ones of Proteobacteria. The observed alterations appear to correlate with high levels of serum endotoxin in a subset of the samples. Network topology analysis indicated that alcohol use is correlated with decreased connectivity of the microbial network, and this alteration is seen even after an extended period of sobriety. We show that the colonic mucosa-associated bacterial microbiome is altered in a subset of alcoholics. The altered microbiota composition is persistent and correlates with endotoxemia in a subgroup of alcoholics.     

Molecular definition of vaginal microbiota in East African commercial sex workers

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV(+)) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV(+) individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV(+) women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV(+) and BV(-) diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV(+) and BV(-). Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV(-) ("normal") phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.     

益生菌对特应性皮炎的防治及机制研究进展

特应性皮炎(atopic dermatitis, AD)是一种以反复发作和严重瘙痒为特征、发病率最高的过敏性皮肤病。AD的致病机制涉及遗传易感性、表皮屏障功能障碍、微生物组失调、免疫反应失衡以及环境等多个因素,而现有治疗用药副作用大、疗效欠佳。目前研究已发现肠道菌群尤其是益生菌在AD中起着重要作用。益生菌能够通过抑制病原菌、增强屏障功能、改善肠道环境和平衡Th1/Th2免疫应答等机制改善AD症状。本文综述了AD患者皮肤及肠道微生态特征,基于AD发病的致病机制和影响因素,系统阐明益生菌缓解AD的机制,以期为益生菌治疗AD及相关皮肤过敏性疾病提供理论支持。     

Selection and identification of anaerobic lactobacilli producing inhibitory compounds against vaginal pathogens

Two strains of Lactobacillus crispatus (15L08 and 21L07) and one strain of Lactobacillus jensenii (5L08) were selected from amongst 100 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization and the production of H2O2 and/or bacteriocin-like compound. All three strains self-aggregated and adhered to vaginal epithelial cells, displacing well-known vaginal pathogens, such as Gardnerella vaginalis and Candida albicans. Lactobacillus crispatus 15L08 was characterized as a potential H2O2 producer. A high level of bacteriocin-like compound was synthesized by L. jensenii 5L08, with a bactericidal mode of action for G. vaginalis, C. albicans and Escherichia coli. However, H2O2-dependent activity alone was not sufficient to inhibit the growth of C. albicans. Simultaneous actions of H2O2 and bacteriocin-like compound produced by lactobacilli may be important for antagonizing pathogenic bacteria. These strains of lactobacilli may be excellent candidates for eventual use as probiotics to restore the normal microbial communities in the vaginal ecosystem.     

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。     

乳杆菌优良菌株的筛选及对小鼠阴道白假丝酵母菌过度增殖干预

目的探讨乳杆菌优良菌株的筛选及其对小鼠阴道白假丝酵母菌过度增殖干预作用。方法对德氏乳杆菌DM8909菌株进行诱变筛选,得到耐抗生素突变株208,以ICR小鼠为实验对象,用甲硝唑进行小鼠阴道菌群脱污染,将白假丝酵母菌接种到小鼠阴道内,构建小鼠阴道白假丝酵母菌过度增殖模型。采用阴道接种德氏乳杆菌DM8909突变株208菌液（2×108CFU/mL）处理,取其阴道冲洗液进行阴道菌群分析,并进行阴道上皮细胞的电镜检查。结果乳杆菌干预后显著清除阴道内的假丝酵母菌,肠杆菌恢复正常水平,与模型组比较,差异有统计学意义（P〈0.05）。结论德氏乳杆菌DM8909抗性菌株208能调整白假丝酵母菌在小鼠阴道内定植,对小鼠阴道上皮细胞有恢复作用。