Response of Gardnerella vaginalis biofilm to 5 days of moxifloxacin treatment

Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.     

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.     

Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

Bacterial vaginosis (BV), a common condition seen in premenopausal women, is associated with preterm labor, pelvic inflammatory disease, and delivery of low birth weight infants. Gardnerella vaginalis is the predominant bacterial species associated with BV, although its exact role in the pathology of BV is unknown. Using immunofluorescence, confocal and transmission electron microscopy, we found that VK2 vaginal epithelial cells take up G. vaginalis after exposure to the bacteria. Confocal microscopy also indicated the presence of internalized G. vaginalis within vaginal epithelial cells obtained from a subject with BV. Using VK2 cells and (35)S labeled bacteria in an invasion assay, we found that a 1 h uptake of G. vaginalis was 21.8-fold higher than heat-killed G. vaginalis, 84-fold compared to Lactobacillus acidophilus and 6.6-fold compared to Lactobacillus crispatus. Internalization was inhibited by pre-exposure of cells to cytochalasin-D. In addition, the cytoskeletal protein vimentin was upregulated in VK2 cells exposed to G. vaginalis, but there was no change in actin cytoskeletal polymerization/rearrangements or vimentin subcellular relocalization post exposure. Cytoskeletal protein modifications could represent a potential mechanism for G. vaginalis mediated internalization by vaginal epithelial cells. Finally, understanding vaginal bacteria/host interactions will allow us to better understand the underlying mechanisms of BV pathogenesis.     

Bacterial vaginosis among a group of married Jordanian women: occurrence and laboratory diagnosis

A total of 310 vaginal swabs collected from a group of married Jordanian women complaining of vaginal discharge were examined for bacterial vaginosis. The scoring system of Nugent for the interpretation of Gram staining was employed. This system revealed the presence of the condition in 29.7% of patients. Results obtained using the scoring system correlated significantly with the detection of clue cells and the scarcity of white blood cells in the vaginal discharge. An inverse relationship was found between bacterial vaginosis and Lactobacillus morphotypes determined by Gram staining. No definite relationship was detected between bacterial vaginosis and the recovery of Gardnerella vaginalis by culture as this organism was isolated from swabs which according to the Nugent criterion were negative for bacterial vaginosis. Bacterial vaginosis among the women investigated was more prevalent than vaginitis caused by Trichomonas vaginalis or yeasts.     

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。     

细菌性阴道病微生态与益生菌

细菌性阴道病(BV)是育龄妇女阴道炎的最常见原因,其特征是栖居在阴道内的乳酸杆菌减少导致阴道菌群平衡失调的复杂变化。用乳酸杆菌替代抗生素是治疗BV的一种有效的治疗措施。乳酸杆菌产生过氧化氢、乳酸、杆菌素能抑制引起BV的细菌生长。多胺在BV的病理机制中有重要作用。本研究拟综述BV患者阴道菌群的变化,免疫防御反应,阴道乳酸杆菌对BV的影响,多胺的意义,益生菌在治疗选择中的作用。     

The range of antagonistic effects of Lactobacillus bacterial strains on etiologic agents of bacterial vaginosis

Bacterial vaginosis is caused by uncontrolled sequential overgrowth of some anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia, Bacteroides spp., Peptostreptococcus spp., Mobiluncus sp. usually occurring in stable numbers in the bacterial flora of healthy women. On the other hand, different species of bacteria belonging to the genus Lactobacillus, most frequently L. plantarum, L. rhamnosus and L. acidophilus, form a group of aerobic bacteria dominating in the same environment. The diversity and density of their populations depend on the age and health conditions. Thanks to their antagonistic and adherence properties bacteria of the genus Lactobacillus can maintain a positive balance role in this ecosystem. The aim of this study was to assess the antagonistic properties of Lactobacillus strains isolated from the vagina of healthy women against most common agents of bacterial vaginosis. It was found that nearly all of the tested Lactobacillus strains exerted distinct antagonistic activity against anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia and Peptostreptococcus anaerobius and quite a number also against Gram-negative rods, while only some of them were able to inhibit Gram-positive aerobic cocci as Enterococcus faecalis or Staphylococcus aureus.     

Functional and phylogenetic characterization of Vaginolysin, the human-specific cytolysin from Gardnerella vaginalis

Pore-forming toxins are essential to the virulence of a wide variety of pathogenic bacteria. Gardnerella vaginalis is a bacterial species associated with bacterial vaginosis (BV) and its significant adverse sequelae, including preterm birth and acquisition of human immunodeficiency virus. G. vaginalis makes a protein toxin that generates host immune responses and has been hypothesized to be involved in the pathogenesis of BV. We demonstrate that G. vaginalis produces a toxin (vaginolysin [VLY]) that is a member of the cholesterol-dependent cytolysin (CDC) family, most closely related to intermedilysin from Streptococcus intermedius. Consistent with this predicted relationship, VLY lyses target cells in a species-specific manner, dependent upon the complement regulatory molecule CD59. In addition to causing erythrocyte lysis, VLY activates the conserved epithelial p38 mitogen-activated protein kinase pathway and induces interleukin-8 production by human epithelial cells. Transfection of human CD59 into nonsusceptible cells renders them sensitive to VLY-mediated lysis. In addition, a single amino acid substitution in the VLY undecapeptide [VLY(P480W)] generates a toxoid that does not form pores, and introduction of the analogous proline residue into another CDC, pneumolysin, significantly decreases its cytolytic activity. Further investigation of the mechanism of action of VLY may improve understanding of the functions of the CDC family as well as diagnosis and therapy for BV.     

Identification and Characterization of Bacterial Vaginosis-Associated Pathogens Using a Comprehensive Cervical-Vaginal Epithelial Coculture Assay

Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.     

Inerolysin, a cholesterol-dependent cytolysin produced by Lactobacillus iners

Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.     

Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.     

Characterization and selection of vaginal Lactobacillus strains for the preparation of vaginal tablets

AIMS: To characterize and select Lactobacillus strains for properties that would make them a good alternative to the use of antibiotics to treat human vaginal infections. METHODS AND RESULTS: Ten Lactobacillus strains belonging to four different Lactobacillus species were analysed for properties relating to mucosal colonization or microbial antagonism (adhesion to human epithelial cells, hydrogen peroxide production, antimicrobial activity towards Gardnerella vaginalis and Candida albicans and coaggregation with pathogens). The involvement of electrostatic interactions and the influence of bacterial metabolic state in the binding of lactobacilli to the cell surface were also studied. Adherence to epithelial cells varied greatly among the Lactobacillus species and among different strains belonging to the same Lactobacillus species. The reduction in surface negative electric charge promoted the binding of several Lactobacillus strains to the cell membrane whereas lyophilization reduced the adhesion capacity of many isolates. The antimicrobial activity of lactobacilli culture supernatant fluids was not directly related to the production of H2O2. CONCLUSIONS: Three strains (Lactobacillus brevis CD2, Lact. salivarius FV2 and Lact. gasseri MB335) showed optimal properties and were, therefore, selected for the preparation of vaginal tablets. The selected strains adhered to epithelial cells displacing vaginal pathogens; they produced high levels of H2O2, coaggregated with pathogens and inhibited the growth of G. vaginalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The dosage formulation developed in this study appears to be a good candidate for the probiotic prophylaxis and treatment of human vaginal infections.     

Lactobacillus strains isolated from the vaginal microbiota of healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell culture

The purpose of this study was to investigate how human vaginal isolates of Lactobacillus acidophilus, Lactobacillus jensenii, Lactobacillus gasseri and Lactobacillus crispatus inhibit the vaginosis-associated pathogens Gardnerella vaginalis and Prevotella bivia. Results show that all the strains in coculture condition reduced the viability of G. vaginalis and P. bivia, but with differing degrees of efficacy. The treatment of G. vaginalis- and P. bivia-infected cultured human cervix epithelial HeLa cells with L. gasseri strain KS120.1 culture or cell-free culture supernatant (CFCS) results in the killing of the pathogens that are adhering to the cells. The mechanism of the killing activity is not attributable to low pH and the presence of lactic acid alone, but rather to the presence of hydrogen peroxide and proteolytic enzyme-resistant compound(s) present in the CFCSs. In addition, coculture of G. vaginalis or P. bivia with L. gasseri KS120.1 culture or KS120.1 bacteria results in inhibition of the adhesion of the pathogens onto HeLa cells.     

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.     

妊娠妇女与无性生活女性阴道分泌物 微生物组学分析

目的探讨妊娠妇女与无性生活女性阴道分泌物中菌群结构差异,为临床女性泌尿生殖系统疾病研究提供可靠的依据。方法运用高通量二代测序技术检测20例妊娠妇女和29例无性生活女性阴道分泌物中微生物;采用秩和检验进行组间显著性差异分析,使用生物信息学软件进行数据处理。结果 49份样本共注释出14个门、23个纲、33个目、28个科、38个属和23个种水平物种。妊娠妇女与无性生活女性阴道分泌物中菌群结构差异显著。妊娠妇女在种水平仅有Atopobium vaginae、Campylobacter ureolyticus、Lactobacillus coleohominis、德氏乳杆菌、瑞氏乳杆菌、惰性乳杆菌、罗伊乳杆菌、白假丝酵母菌和近平滑假丝酵母菌共9个物种。结论妊娠能引起女性阴道分泌物菌群结构发生改变,且菌种数量明显减少。Lactobacillus coleohominis和德氏乳杆菌可能在维持妊娠妇女阴道菌群平衡和自净作用中起到关键性作用。     

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.     

The use of the biotherapeutic agent Zhlemik for correcting the microflora in bacterial vaginosis

The vaginal microflora in 11 women suffering with bacterial vaginosis was corrected by BTA "Zlemik" contained highly adhesive strain Lactobacillus. BTA was administered intravaginal in dose 1 suppositoria at night during 15 days. It was established in majority cases that clinical symptoms and discharges have disappeared. The value of pH has decreased from 5.6-6.2 to 4.6-4.8. After biotherapeutic therapy significantly increased of the levels of Lactobacillus and percentage of the presence Bifidobacterium. The number of non-spore-forming obligatory anaerobic bacteria, Streptococcus and Gardnerella decreased in 10 and 100 times respectively.     

Electron microscopy of adhesive interactions between Gardnerella vaginalis and vaginal epithelial cells, McCoy cells and human red blood cells

Exfoliated vaginal epithelial cells with attached bacteria, termed 'clue cells', which were procured from a patient with non-specific vaginitis, were stained with ruthenium red and examined by transmission electron microscopy. The attached bacteria appeared to adhere by means of an outer fibrillar coat. An epithelial tissue culture cell line (McCoy) and human red blood cells to which strains of Gardnerella vaginalis attached were similarly examined. The adherence of G. vaginalis to the epithelial cell line appeared to be mediated by an outer fibrillar coat while adherence to red cells appeared to be mediated by fimbriae. Transmission electron microscopy was performed on the Gardnerella strains used. Thin sections of tissue-culture-adherent strains revealed a dense outer fibrillar coat whereas the surface of the haemagglutinating strains showed fine fimbriae. Negative staining of haemagglutinating strains demonstrated fimbriae on a minority of organisms.     

妇肤康喷雾剂对阴道常见微生物增殖影响的体外研究

目的体外了解妇肤康喷雾剂溶液对阴道常见的益生乳杆菌和致病菌的影响,为阴道感染疾病提供药物应用的理论依据,指导临床用药。方法对从中国健康妇女阴道分离的乳杆菌、ATCC阴道来源的加德纳菌和白色念珠菌进行体外培养,以中药制剂妇肤康喷雾剂作为培养基,通过绘制生长曲线,评价妇肤康溶液对阴道常见微生物的影响。结果妇肤康实验组中,乳杆菌和白色念珠菌生长曲线随着时间延长轻微下降。加德纳菌在15h后可完全杀灭。结论体外培养时,妇肤康溶液轻微抑制乳杆菌生长,其能够抑制白色念珠菌生长,可以杀灭加德纳菌。