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.     

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.     

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.     

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.     

Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina

The vagina has been increasingly viewed as an "ecosystem" whose normal microflora help protect it from invading pathogens, including those that cause urinary tract infections and sexually transmitted diseases. We tested new strains of lactobacilli as potential probiotics for maintenance of urogenital tract health, as well as prevention and therapy of urogenital infections. A strain of lactobacilli isolated from the vagina of nonpregnant, healthy, premenopausal women was identified as Lactobacillus rhamnosus L60 by 16S rDNA sequence homology. L60 was evaluated for antimicrobial activity, in vitro antibiotic resistance, autoaggregation, surface hydrophobicity, co-aggregation with other bacterial species, hydrogen peroxide (H(2)O(2)) production, and bacterial adherence. It displayed a wide spectrum of antimicrobial activity against urogenital pathogens, and resistance to antibiotics commonly prescribed for infections caused by these pathogens. L60 produced H(2)O(2), adhered to vaginal epithelial cells, co-aggregated with Escherichia coli and Candida albicans, and displayed self-aggregation. In view of these characteristics, L60 is considered a potential probiotic, and will be further evaluated for preventive and therapeutic application locally in the vaginal tract.     

阴道微生态与宫颈疾病发生发展的相关性研究

摘要：微生态学是近年新兴的学科,女性生殖道是一个重要的微生态区域。生殖道微生态体系是由正常的解剖结构、生殖道各种菌群、周期性内分泌变化、局部免疫系统四个部分组成。随着阴道微生态平衡理论的提出和发展,阴道微生态和HPV感染、宫颈上皮内瘤变、宫颈癌的内在关联引起了极大的重视。阴道微生态与宫颈疾病的发生发展相关,宫颈病变影响了阴道微生态环境,长期的阴道菌群失调也会反作用于宫颈,导致宫颈的病变加重甚至恶化,如此形成恶性循环。因此阴道微生态对宫颈疾病的诊断、治疗和预防将具有重要的临床意义。     

Identification method based on PCR combined with automated ribotyping for tracking probiotic Lactobacillus strains colonizing the human gut and vagina

AIMS: A molecular methodology based on PCR-associated automated ribotyping was developed to specifically detect the Lactobacillus strains of two probiotic products (an orally administered lyophilized preparation and vaginal tablets) in human faeces and vaginal swabs. METHODS AND RESULTS: The 16S-23S rDNA sequences and the ribotype profiles of the probiotic lactobacilli were characterized and new species-specific primer sets were designed. The identification of faecal and vaginal lactobacilli isolated from subjects administered with the probiotic products was performed by using PCR with species-specific primers followed by strain-specific automated ribotyping. CONCLUSIONS: The PCR-ribotyping identification allowed to study the colonization patterns of the probiotic lactobacilli in the human gut and vagina evidencing the strains with the best survival capability. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed molecular method represents a powerful tool of strain-specific identification, useful for differentiating exogenous from indigenous strains in any microbial ecosystem and for rationally choosing probiotic bacteria with the best chance of survival in the host.     

Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora

Background  

Despite their antimicrobial potential, vaginal lactobacilli often fail to retain dominance, resulting in overgrowth of the vagina by other bacteria, as observed with bacterial vaginosis. It remains elusive however to what extent interindividual differences in vaginal Lactobacillus community composition determine the stability of this microflora. In a prospective cohort of pregnant women we studied the stability of the normal vaginal microflora (assessed on Gram stain) as a function of the presence of the vaginal Lactobacillus index species (determined through culture and molecular analysis with tRFLP).     

女性生殖道微生态评价研究进展

女性生殖道微生态体系由阴道内的微生态菌群、机体内分泌调节功能和解剖结构共同组成。建立客观全面的评价体系对生殖健康、疾病诊断与治疗预后判定、以及生殖道感染预防技术的微生态环境影响的评价等都具有重要意义。目前对于女性生殖道微生态评价的方法主要有常规理化生化方法、微生物培养与鉴定、以及分子生物学核酸或脂肪酸等生物大分子标志物检测等。本文主要对近年来女性生殖道微生态评价的研究进展,特别是对女性生殖道的绝对优势菌群乳酸杆菌的功能研究加以综述,并对本领域未来的研究方向进行了展望。     

34例胎膜早破孕妇妊娠晚期阴道微生态及免疫因子变化状况分析

目的：胎膜早破是一种常见但发生机制十分复杂的妊娠并发症。在所有的早产儿中,孕妇胎膜早破的比例高达33％。本研究针对胎膜早破产妇妊娠晚期阴道微生态及免疫因子的变化情况,分析阴道内茵群失调或局部免疫反应与胎膜早破的关系,为临床研究提供实验数据。方法：回顾性分析我院于2010年7月-2013年3月收治的34例胎膜早破孕妇的临床资料,同时选取于我院进行健康体检的妇女及产前检查的健康孕妇各34例作为对照。采集所有研究对象的阴道分泌物,对比分析各组样本中病原微生物的分布及免疫因子的浓度。结果：胎膜早破组乳酸杆菌的检出率要明显低于健康妇女组与健康孕妇组（79．4％VS70．6％VS34．1,X2=8．438,P〈0．05）,三组研究对象其他茵属分布的差异比较无统计学意义（P〉0．05）;胎膜早破组阴道分泌物IL-6浓度与TNF．仪浓度要显著高于健康妇女组与健康孕妇组（P〈0．05）。结论：胎膜早破产妇阴道内的菌群分布与健康女性及孕妇有所不同,阴道内茵群失调及局部的免疫反应可能会导致胎膜旱破的发生。     

Normal vaginal flora in chimpanzees (Pan troglodytes): qualitative and quantitative study

Lactobacilli are the predominant microorganisms in the vaginal flora of human beings, and are known to play an important role in protecting them from genital infections. On the other hand, the composition of the vaginal flora differs among laboratory animal species, and lactobacilli are not the predominant vaginal microorganism in many laboratory animals. We speculated that the vaginal flora of chimpanzees would be more similar to those of human beings than to those of other animal species, because chimpanzees are phylogenetically close to human beings, and their reproductive physiology is similar to that of human beings. To clarify our speculation, we examined the development of the vaginal flora in chimpanzees (Pan troglodytes). Streptococci, lactobacilli, and members of the family Bacteroidaceae were the most predominant bacteria in the vagina of mature chimpanzees (9 to 22 years old). During development of the vaginal flora of chimpanzees, the total number of bacteria increased with age and reached a plateau just before sexual maturity (5 to 7 years of age; juvenile period). Lactobacilli were already one of the predominant bacteria before sexual maturity. In mature chimpanzees, the total number of bacteria (aerobes and anaerobes) in the vagina was highest during the swelling phase of the menstrual cycle. During the swelling phase in mature chimpanzees, streptococci, lactobacilli, and Bacteroidaceae were the most frequently isolated (100%) organisms, and the total number of organisms recovered from vaginal specimens from these three groups was the highest. In mature chimpanzees in which the number of bacteria was the highest, lactobacilli were the predominant bacteria. Taken together, these results suggest that these three bacterial groups (streptococci, lactobacilli, and Bacteroidaceae) are indigenous to the vagina of chimpanzees, and chimpanzees would be the most suitable laboratory animals for studying the role of lactobacilli in the vagina of human beings.     

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.     

Changes in vaginal microbiocenosis in patients with chronic pelvic inflammatory diseases following antibiotic therapy

The species composition of the vaginal microorganisms in healthy women and in patients with chronic pelvic inflammatory diseases before and after treatment in a gynecological hospital was studied. The study revealed that antibiotic therapy did not lead to complete clinical convalescence. During bacteriological investigation of patients changes in vaginal microbiocenosis, manifested by a decreased number of microbial species, an increased proportion of Escherichia coli, the occurrence of Staphylococcus aureus, a decreased number of Lactobacillus ssp., were observed. Antibiotic therapy aggravated the dysbiotic microbial picture of the vagina.     

Vaginal microflora in girls of prepuberal age

The bacteriological study of vaginal microflora in 24 clinically healthy girls aged 7-8 years was carried out. Two criteria of normal vaginal biocenosis in girls of prepuberal age were proposed. The proportion of hemolytic bacteria in the vagina of a girl of prepuberal age could reach 100% of all microflora, provided the total bacterial count (TBC) exceed average values. A high proportion of hemolytic bacteria in the vaginal microflora of girls of prepuberal age in combination with a high level of TBC (even with TBC not exceeding the upper limit of its normal value) could probably by regarded as a border state. No fungi of the genus Candida should be isolated from vagina in girls of prepuberal age. Facultative anaerobic microflora was represented mainly by Staphylococcus epidermidis and S. saprophyticus. In 64.3 +/- 12.8% of cases bifidobacteria were detected in the girls vagina.     

Antilactoferrin activity of microorganisms

The detection rate of the antilactoferrin sign and the level of its manifestation in 165 strains of different microbial species, isolated from patients with inflammatory diseases and intestinal dysbacteriosis, were analyzed. The detection rate of antilactoferrin activity was 43 - 90% for Escherichia coli strains, 20 - 86% for Staphylococcus aureus, 60 - 100% for Klebsiella pneumoniae, 76 - 78% for Candida albicans, isolated from different biotopes of man. Most frequently and with high levels of manifestation this sign was registered in strains isolated from the reproductive tract of women. The detection rate of this sign and the level of its manifestation in bacteria of the vaginal and cervical microbiocenosis, isolated from patients, were higher in comparison with healthy persons. The inverse dependence between the level of the antilactoferrin activity of microflora and the content of lactoferrin during the inflammatory process in women was established.     

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.     

乳杆菌在阴道微生态中生存状态的研究进展

乳杆菌是维持阴道微生态平衡的重要菌群,其通过粘附定植,免疫调节,分泌细菌素、类细菌素物质等多种途径抑制其它微生物的生长。同时乳杆菌自身的生存也容易受到如阴道微环境,性生活等多种因素的影响,导致阴道微生态失衡,诱发多种妇科疾病。     

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。     

乳酸菌与阴道疾病关系的研究进展

乳酸菌是阴道菌群中的优势菌群,能够分解糖原产生乳酸、过氧化氢及多种抗微生物因子,可从多方面调节阴道内需氧菌和厌氧菌的比例,维持着阴道内的菌群平衡。当这种平衡被其他各种因素打破时,便会引起阴道疾病,通过补充活性乳酸菌可以达到对阴道疾病的预防和治疗。本研究概述了六类常见的阴道疾病,并对近年来乳酸菌治疗此类疾病的机制极其研究进展进行了阐述。