Binding of catalase by Gardnerella vaginalis

Previous work has demonstrated that Gardnerella vaginalis can utilize catalase as a sole source of iron. In this study, the interaction between G. vaginalis cells and catalase was investigated. G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled catalase using a solid phase dot blot assay. An increase in catalase binding was observed from cells grown under iron-restrictive conditions. Western blot analysis of G. vaginalis proteins resulted in the detection of a putative catalase-binding protein with an estimated molecular mass of 128 kDa. The 128-kDa catalase-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis suggesting this protein may be surface-exposed.     

Haemagglutination and tissue culture adhesion of Gardnerella vaginalis

Six strains of Gardnerella vaginalis were studied to examine the adhesin-receptor mechanism involved in their attachment to human red blood cells and an epithelial tissue culture cell line (McCoy). The adhesins involved in the attachment of the bacteria to each of these cells were proteinaceous but showed marked differences after various chemical or physical treatments, indicating that separate adhesins were present. Haemagglutinating strains were more hydrophobic than tissue-culture-adherent strains. Haemagglutination of human red blood cells by strains of G. vaginalis was inhibited by galactose, lactose, N-acetylneuraminic acid and phosphatidylserine. In contrast, the tissue-culture adherence of strains was not inhibited by these substances.     

Prevalence of Gardnerella vaginalis: an estimate

To assess the prevalence of Gardnerella vaginalis in the community 300 women aged 16-59 were randomly selected from a general practice''s age-sex register and invited to attend for a health check. Out of 282 women who were eligible to attend, 192 did so. They were asked whether they had any vaginal symptoms, and swabs were taken from 182 women for culture for G vaginalis. Sixty women were positive for G vaginalis, of whom 26 had symptoms.Infections with G vaginalis may be present in women who have no symptoms. By careful questioning, examination, and side room testing general practitioners may be able to diagnose these infections in such women consulting them for other reasons.     

Identification of a Gardnerella vaginalis Hemoglobin-Binding Protein

Previous studies have shown that Gardnerella vaginalis can utilize human hemoglobin as a sole source of iron. In this study, the interaction between human hemoglobin and G. vaginalis cells was investigated. With a solid phase dot blot assay, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human hemoglobin. A human hemoglobin-binding protein with an estimated molecular weight of 124 kilodaltons (kDa) was detected by Western blot analysis of G. vaginalis proteins. The hemoglobin-binding activity of this protein was found to be heat stable and was observed in G. vaginalis cells grown under iron-restrictive and iron-replete conditions. The 124-kDa hemoglobin-binding protein was not detected from intact G. vaginalis cells treated with trypsin prior to Western blot analysis, suggesting that this protein was surface exposed. Received: 26 June 2000 / Accepted: 21 July 2000     

Screening of Compounds against Gardnerella vaginalis Biofilms

Bacterial vaginosis (BV) is a common infection in reproductive age woman and is characterized by dysbiosis of the healthy vaginal flora which is dominated by Lactobacilli, followed by growth of bacteria like Gardnerella vaginalis. The ability of G. vaginalis to form biofilms contributes to the high rates of recurrence that are typical for BV and which unfortunately make repeated antibiotic therapy inevitable. Here we developed a biofilm model for G. vaginalis and screened a large spectrum of compounds for their ability to prevent biofilm formation and to resolve an existing G. vaginalis biofilm. The antibiotics metronidazole and tobramycin were highly effective in preventing biofilm formation, but had no effect on an established biofilm. The application of the amphoteric tenside sodium cocoamphoacetate (SCAA) led to disintegration of existing biofilms, reducing biomass by 51% and viability by 61% and it was able to increase the effect of metronidazole by 40% (biomass) and 61% (viability). Our data show that attacking the biofilm and the bacterial cells by the combination of an amphoteric tenside with the antibiotic metronidazole might be a useful strategy against BV.     

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

Abstract The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al, 1991) FEBS Lett. 283, 317–320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 Å and 28 Å in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K⁺ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca²⁺ concentration, suggesting a Ca²⁺ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca²⁺ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.     

Detection of Gardnerella vaginalis on vaginal smears by immunofluorescence

An indirect fluorescence antibody (IFA) test was developed for the detection of Gardnerella vaginalis. Antisera were prepared in rabbits by using five strains of G. vaginalis. A pool of the antisera was tested for specificity with a variety of isolates known to colonize the human vagina and (or) morphologically resemble G. vaginalis. Six heterologous bacterial isolates reacted with the pooled antiserum at dilutions of 1:10, but none reacted at the working dilution of 1:200. Vaginal swab specimens were collected from symptomatic and asymptomatic patients in order to further evaluate the IFA procedure. The presence of G. vaginalis in the specimens was determined both by culture and by the IFA procedure. Absorbed antisera reacted with all isolates of G. vaginalis tested. In a clinical trial the IFA procedure detected the presence of G. vaginalis in smears from 23 (24.2%) of the patients with nonspecific vaginitis (NSV), from 22 (29.8%) of the asymptomatic individuals tested, and from 3 patients with vaginitis other than NSV. The presence of G. vaginalis in smears as detected by the IFA procedure was confirmed by cultures in all cases using Vaginalis agar supplemented with colistin and nalidixic acid (V-CNA). It is suggested that the IFA procedure may be of use in conjunction with V-CNA in epidemiological studies of the carriage and transmission of G. vaginalis in human populations. It appears that the IFA procedure, at least in our hands, is a useful test for the rapid detection of G. vaginalis even when this microorganism is not the predominant colonizer of the human vagina.     

阴道液中唾液酸酶活性、阴道加德纳菌及其IgA水平的相关性研究

目的探讨阴道液中唾液酸酶活性、阴道加德纳菌及抗阴道加德纳菌的溶血素(anti-Gvh)IgA水平在细菌性阴道病(BV)的相关性。方法对15例健康人和60例临床诊断为BV的患者,取阴道分泌物分别进行唾液酸酶活性和anti-Gvh IgA水平测定及阴道加德纳菌(Gv)分离培养。唾液酸酶活性利用从底物-5溴-4氯-3吲哚基-α-D-N乙酰基神经氨酸(X-Neu5Ac)转化而得到的甲氧基苯酚的纳摩尔数来表示;anti-Gvh IgA通过ELISA法测定。结果BV组Gv活菌数(6.96 log CFU/g)显著高于健康对照组(2.58 log CFU/g)(P<0.01)。BV组anti-GvhIgA(238.0±220.6)显著高于健康对照组(175.0±40.16)(P<0.01)。BV组Gv分离率(86.7%)显著高于健康对照组(33.3%)(P<0.01)。45例临床诊断为BV的患者中,其中26例Gvh IgA(+),19例Gvh IgA(-);Gvh IgA(-)组阴道液中唾液酸酶活性(5.00±1.29)显著高于Gvh IgA(+)组(1.58±1.22)(P<0.01),2组的Gv的分离率和活菌数差异却没有显著性(P>0.05)。结论BV患者阴道内高活性的唾液酸酶与低水平anti-Gvh IgA和黏膜IgA破坏程度具有关联性。     

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.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

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.     

Oleate lipase activity in Gardnerella vaginalis and reconsideration of existing biotype schemes

Background  

Gardnerella vaginalis is a facultative gram positive organism that requires subculture every 1–2 days to maintain viability. It has been linked with bacterial vaginosis (BV), a syndrome that has been associated with increased risk for preterm delivery, pelvic inflammatory disease and HIV acquisition. About 10% of the G. vaginalis isolates have been reported to produce sialidase, but there have not been any studies relating sialidase production and biotype. Sialidase activity is dramatically increased in the vaginal fluid of women with BV and bacterial sialidases have been shown to increase the infectivity of HIV in vitro. There are 8 different biotypes of G. vaginalis. Biotypes 1–4 produce lipase and were reported to be associated with BV and the association of these biotypes with BV is under dispute. Other studies have demonstrated that G. vaginalis biotype 1 can stimulate HIV-1 production. Because of the discrepancies in the literature we compared the methods used to biotype G. vaginalis and investigated the relationship of biotype and sialidase production.     

Contribution of Gardnerella vaginalis to vaginitis in a general practice.

In a study of 154 adult women who presented to their general practitioner with vaginal symptoms 30 (20%) had Gardnerella vaginalis on its own and 51 (33%) had G vaginalis in combination with anaerobes or known pathogens. Thirty one (20%) patients were culture negative. Those who were culture negative had fewer symptoms and signs of vaginitis than those with G vaginalis alone or G vaginalis plus anaerobes. Those with known pathogens had more symptoms and signs than those with G vaginalis alone or G vaginalis plus anaerobes. Those with known pathogens plus G vaginalis had the most severe signs and symptoms of vaginitis. It is concluded that G vaginalis can cause vaginitis on its own, and it makes vaginitis worse when present with other organisms. G vaginalis was also found in 30 (21%) of the 138 control patients who, although they presented "asymptomatically," had worse signs than control patients without G vaginalis. It seems that G vaginalis can occur in a spectrum ranging from the uncomplaining patient to those with severe vaginitis.     

Zinc Lactate and Sapindin Act Synergistically with Lactocin 160 Against Gardnerella vaginalis

Lactocin 160 is a vaginal probiotic-derived bacteriocin shown to selectively inhibit the growth of Gardenerella vaginalis and some other pathogens commonly associated with bacterial vaginosis. The natural origin of this peptide, its safety, and selective antimicrobial properties make it a promising candidate for successful treatment and prophylaxis of bacterial vaginosis (BV). This study evaluated interactions between lactocin 160 and four other natural antimicrobials in the ability to inhibit G. vaginalis. We report that zinc lactate and soapnut extract act synergistically with lactocin 160 against this pathogen and therefore have a potential to be successfully used as the components of the multiple-hurdle antimicrobial formulation for the treatment of BV.     

Loop‐mediated isothermal amplification for the rapid detection of Gardnerella vaginalis

The aim of this study was to develop a method for the rapid detection of Gardnerella vaginalis, which is proposed to play a key role in the pathogenesis of bacterial vaginosis. Specific loop‐mediated isothermal amplification (LAMP) primers were designed and used to detect target DNA within 45 min under isothermal conditions. Comparative screening indicated that the LAMP assay is superior to PCR in terms of rapidity, and is equivalent in sensitivity and specificity. This LAMP assay can be used for rapid screening and detection of G. vaginalis in vaginal samples; the limit of detection is 10 fg DNA.

     

Fatty acid,polar lipid and wall amino acid composition of Gardnerella vaginalis

Representative strains of Gardnerella vaginalis were degraded using both an alkaline and an acid methanolysis and the fatty acid methyl esters released examined by thin-layer and gas chromatography. The profiles obtained were both qualitatively and quantitatively similar and were comprised of straight chain saturated and unsaturated non-hydroxylated fatty acids with hexadecanoic acid (16:0) and octadecenoic acid (18:1) the major components. All of the strains contained very characteristic polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, five partially identified glycolipids and an uncharacterised phospholipid. Analyses of wall amino acid preparations using gas chromatography showed that Gardnerella vaginalis strains contain major amounts of alanine, glycine, glutamic acid and lysine. The chemical data support the integrity of the genus Gardnerella.     

Detection of a species-specific antigen of Gardnerella vaginalis by Western blot analysis

Western blot analysis was used to identify antigenic components of Gardnerella vaginalis. Polypeptides bound to nitrocellulose membranes were probed with murine antisera raised to two strains of G. vaginalis, and antibody-antigen complexes were detected with 125I-labelled antimouse immunoglobulin followed by autoradiography. Although there was inter-strain variation in immunogenic polypeptide profiles, all 23 strains of G. vaginalis examined contained a common antigen of molecular mass 41 kDa. This antigen was not found in any of six other bacterial genera.     

阴道加德纳菌检出率及唾液酸酶A基因携带与细菌性阴道病的关系

目的探究阴道加德纳菌(Gardnerella vaginalis)检出率及唾液酸酶A基因携带与细菌性阴道病(BV)的关系。方法选择2017年1月至2019年8月确诊的BV患者82例作为BV组,并随机选择同时期健康女性82例作为健康组,比较2组人群G. vaginalis检出率和唾液酸酶A基因携带情况,相关统计学资料分析其对BV发生的影响。结果BV组人群G. vaginalis阳性检出率高于健康组(χ²=11.511,P<0.05)。BV组人群共检出G. vaginalis 1、2、3、4、5、6和7型,其中2型占比最高;BV组人群G. vaginalis 2、3、4型占比率高于健康组(χ²=4.148,17.009,9.973,均P<0.05)。BV组人群唾液酸酶A基因携带率高于健康组(χ²=39.234,P<0.05)。较健康组人群,BV组PCR DGGE宽度更窄,肠道菌群条带数少(t=9.217,P<0.05)。结论G. vaginalis检出率和唾液酸酶A基因携带情况与BV发生相关,有待成为相关生物学治疗靶点。     

Comparative Genomics of Gardnerella vaginalis Strains Reveals Substantial Differences in Metabolic and Virulence Potential

Background

Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9.

Principal Findings

Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species.

Conclusions

Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential, although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G. vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism.