Influence of anaerobic conditions on vaginal microbiota recovery from bacterial vaginosis patients

Bacterial vaginosis (BV) is one of the most common infections in women of reproductive age. Clinical studies have shown an association among BV and abnormal pregnancy, pelvic inflammatory disease and increased risk of sexually transmitted infections, including HIV. This disorder was first described in 1914 by Curtis as a “white discharge” syndrome and despite the decades of research we have only limited, and clearly not conclusive, evidence of microbial cause of BV, mechanism of disease and effective treatment. The development of molecular techniques such as Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing produced a clearer picture of the complexities of the vaginal microbiota. It has also become more apparent that none of the microorganisms already isolated from the vagina are likely to be the sole pathogen responsible for BV. Thus, improved knowledge of the relationship between different species of bacteria and their host is critical to a better understanding of both women’s health and illness. In order to comprehend the dynamic interaction between bacteria–bacteria and bacteria–host it becomes necessary to isolate bacteria from vaginal samples and to use them in in vitro and in vivo assays. The human vagina has a very specific environment regarding pH, nutrient availability and oxygen tension, being populated by a large range of bacteria from facultative to obligated anaerobic species. To isolate bacteria from such a complex niche, rich media should be used in order to promote the growth of different species. By using Columbia Agar (CBA) with 5% horse blood and by incubating at 37°C in the presence of 5%, 10% CO or in anaerobic condition generated by AnaeroGenTM (Oxoid), we were able to recover different bacteria species, from each of the anaerobic conditions tested, from BV vaginal samples, including some species never reported, as far as we know, such as Brevibacterium ravenspurgense, Corynebacterium tuscaniense, Klebsiella variicola, Nosocomiicoccus ampullae, Staphylococcus warneri and Bacillus firmus. Despite the development of molecular methods and their unquestionable advantages in characterising the vaginal microbiota, classical bacteriology will nevertheless be required to elucidate the etiology of BV since the isolation of the pathogenic agent will be always required. Our findings revealed that slight differences in anaerobic conditions were crucial for the isolation of novel BV-associated organisms. Further in vitro studies of all the isolated bacteria will promote a better understanding of the dynamics of their interaction and will potentially reveal how BV develops and influences other sexual transmitted diseases.(undefined

Lactobacilli and its metabolites as potential probiotics against Gardnerella vaginalis

Bacterial vaginosis (BV) is one of the most common gynecological disorder affecting women in reproductive age. This condition poses a significant health risk because it predisposes women to abnormal pregnancy, pelvic inflammatory disease and an increased risk of sexual transmitted infections, including HIV [1]. Although BV etiology remains unknown, it is characterized by a decrease in vaginal lactobacilli and a proliferation of anaerobes [1, 2]. Despite of the richness and diversity found into BV anaerobes, Gardnerella vaginalis is present in over 90% of the pathologic cases and several studies report its potential as the main etiological candidate [3, 4, 5]. Current BV treatment is strictly based in antibiotic therapy resulting in an increased resistance of BV anaerobes, along with severe reduction of the healthy lactobacilli strains in the vaginal epithelium. Therefore, a more appropriate treatment is required, aiming to decrease G. vaginalis and also to promote the lactobacilli re-colonization in BV patients [6]. Previous studies showed the potential of lactobacilli in preventing vaginal colonization by pathogens, and thus the development of infections, by different mechanisms including auto-aggregation, co-aggregation with pathogenic microorganisms, and adhesion to epithelial cells and/or by producing some metabolites (such as lactic acid, hydrogen peroxide, bacteriocins and biosurfactants) that may act as growth inhibitors or anti-adhesive agents [7]. These probiotic properties have inspired new treatment strategies for vaginal infection. One alternative therapy for BV is the re-colonisation of vagina with lactobacilli species [6]. However, the major gap in this option resides in the choice of the most suitable lactobacillus species. Our goal was to evaluate the probiotic potential of intra and extracellular metabolites from a broad range of lactobacilli strains against several G. vaginalis strains. To accomplish our goal, we isolated 60 vaginal lactobacilli strains from healthy women and performed a screening by an agar spot test against 9 G. vaginalis strains (3 from culture collection and 6 clinical isolates from women with BV) in order to selected the most interesting probiotic candidates. We also included more than 30 culture collection lactobacilli strains. For the best candidates we determined the minimum inhibitory concentration (MIC) and identified the lactobacilli metabolites responsible for G. vaginalis growth inhibition. Our results showed that certain latobacilli metabolites were able to inhibit G. vaginalis growth. In spite of the detection of intracellular biosurfactants in some strains, they were unable to reduce G. vaginalis proliferation. On the other hand, extracellular products of some lactobacilli showed a significant effect on G. vaginalis growth. Overall, from the 90 lactobacilli strains tested, only 4 culture collection and 3 clinical isolate lactobacilli strains exhibited a broad probiotic activity against all the G. vaginalis strains tested. However, only culture collection strains were able to reduce G. vaginalis strains growth to 20-30% in MIC assays, illustrating an efficient probiotic activity by itself. Interestingly, none of these lactobacilli strains belong to the vaginal microflora, revealing a more pronounced probiotic activity than any of those vaginal isolate lactobacilli tested. Therefore, our data suggests the existence of non-vaginal lactobacilli strains possessing probiotic activity against numerous G. vaginalis strains, which may contribute for a new and more effective BV treatment than the currently used therapies

Isolation of Gardnerella vaginalis from BV patients and healthy women : analysis of virulence through adherence, biofilm formation and cytotoxicity assays

Bacterial vaginosis (BV) is one of the most common gynaecological disorder affecting women in the reproductive age. Microbiological analysis of BV has shown Gardnerella vaginalis to be the most frequent organism in BV. However, G. vaginalis colonization do not always lead to BV. This raised the question whether there are pathogenic and commensal lineages within this species. In an effort to understand the differences between G. vaginalis strains, we performed in vitro assays to compare virulence properties of recently isolated 14 G. vaginalis strains from Portuguese women with and without BV. G. vaginalis strains were characterised for their initial adhesion ability to a monolayer of HeLa cells by incubating the bacteria with this monolayer and quantifying the adhesion by staining with DAPI and fluorescence microscopy. These assays revealed that the BV isolates of G. vaginalis had a stronger initial adhesion capability than non-BV isolates. The biofilm-forming capacity was then assessed by allowing each of the strains to form biofilms under anaerobic conditions for 48 hours and using different growth media. It was possible to observe that BV isolates tend to growth preferentially as biofilms while non-BV isolates had a lower intrinsic tendency towards biofilm formation. In addition, BV isolates of G. vaginalis displayed robust cytotoxicity in the epithelial cells after 3 hours in the contact with a monolayer of HeLa cells. Thus, this study outlines two distinct variants of G. vaginalis, one apparently commensal and one pathogenic, and presents evidence for disparate virulence potentials

Gardnerella vaginalis virulence potential outcompetes with 30 other microorganisms isolated from BV patients

Bacterial vaginosis (BV) is the most common vaginal disorder affecting millions of women every year, and is usually associated with several adverse health outcomes, including preterm birth and acquisition of sexually transmitted diseases. However, the etiology of BV is still under debate. Recently, new fastidious anaerobic bacteria have been associated with BV, but there are very few studies that comprehensively evaluate the virulence potential of these microorganisms, mainly due to difficult growth conditions. However, in order to find answers to some of the questions related to BV, isolation and characterization of such bacteria will be required. In this work, samples of vaginal exudate from BV women were collected and 31 different microorganisms were isolated, including 6 novel species never described in BV before. Each microorganism was characterized for their ability to adhere to HeLa epithelial cells and cause cytopathogenic changes; their intrinsic biofilm-forming capability; and finally their antimicrobial susceptibility tests for antibiotics commonly used in the treatment of BV. Despite the strong evidence that G. vaginalis outcompeted the other species in the defined virulence assays, our results also demonstrate that other bacteria showed significant biofilm-forming capability, but not initial adhesion, such as Actinomyces turicensis and Corynebacterium tuscaniense. This work supports the evidence that G. vaginalis is the main colonizer in multi-species related BV and further describes novel microorganisms with enhanced virulence potential. Finally, this is the first characterization of Portuguese microbiota associated with BV

Gardnerella vaginalis outcompetes 29 other bacterial species isolated from BV patients in an in vitro biofilm formation model

Despite the worldwide prevalence of bacterial vaginosis (BV), its etiology is still unknown. Although BV has been associated with the presence of biofilm, the ability of BV-associated bacteria to form biofilms is still largely unknown. Here, we isolated 30 BV-associated species and characterized their virulence, using an in vitro biofilm formation model. Our data suggests that Gardnerella vaginalis had the highest virulence potential, as defined by higher initial adhesion and cytotoxicity of epithelial cells, as well as the greater propensity to form a biofilm. Interestingly, we also demonstrated that most of the BV-associated bacteria had a tendency to grow as biofilms.This work was supported by the European Union (FEDER/COMPETE funds) and the Fundacao para a Ciencia e a Tecnologia (reference FCOMP-01-0124-FEDER-008991 [PTDC/BIA-MIC/098228/2008] and RECI/EBB-EBI/0179/2012 [FCOMP-01-0124-FEDER-027462])

In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, atopobium vaginae and lactobacillus spp

Bacterial vaginosis (BV) is a common pathology of women in reproductive age that can lead to serious health complications, and is associated with shifts in the normal microflora from predominance of Lactobacillus spp. to a proliferation of other anaerobes such as G. vaginalis and A vaginae, which can be detected by PCR. The optimal PCR pathogen detection assay relies mainly on the specificity and sensitivity of the primers used. Findings Here we demonstrate that in silico analytical testing of primer specificity is not a synonym to in vitro analytical specificity by testing a range of published and newly designed primers with both techniques for the detection of BV-associated microorganisms. Conclusions By testing primer in vitro specificity with a sufficient range of bacterial strains, we were able to design primers with higher specificity and sensitivity. Also by comparing the results obtained for the newly designed primers with other previously published primers, we confirmed that in silico analysis is not sufficient to predict in vitro specificity. As such care must be taken when choosing the primers for a detection assay.(undefined

Comparison of vaginal microbiota fingerprints from healthy and bacterial vaginosis-positive portuguese women

Bacterial Vaginosis (BV) is a common disease in women of reproductive age and is characterized by the substitution of Lactobacillus species,which are predominant in the normal vaginal microbiota,by rapidly proliferating anaerobic bacteria, particularly Gardnerellavaginalis. The aim of this study was to study microbial communities’ structure in the vaginal microbiota of healthy and BV-positive Portuguese women. To this end, DNA obtained from vaginal samples of 22 BV-negative and 19 BV-positive women was analyzed using a PCR-DGGE approach.Total bacterial communities were amplified using general 16S rRNA gene primers. Group-specific primers were also used targeting Lactobacillus and Bifidobacterium genera and G.vaginalis. DGGE profiles were compared using the BioNumericsTM software package (Applied Maths, Belgium). Similarity between DGGE profiles was determined by calculating similarity indices of the densitometric curves of the compared profiles, using the Dice product-moment correlation. Different DGGE profiles could be obtained for BV-positive and BV-negative samples and this was verified for all primers sets utilized, suggesting that alteration of microbial community structure of BV-positive and -negative samples could be detected by PCR-DGGE. DGGE profiles obtained from samples of BV-positive women were more diverse that the ones from healthy women (as determined by a higher number of DGGE bands). The analysis of the standard electrophoretic bands for bacteria reveals an intrinsic diversity even within the two groups studied: similarities in bacterial DGGE profiles vary between 14- 78% and 47-100% in BV-positive and BV-negative samples, respectively. Among the 19 BV-positive women studied 18 were colonized with G. vaginalis. G.vaginalis was not detected in any of the healthy women samples. The analysis of Lactobacillus communities revealed a higher diversity in BV-negative women than in BV-positive ones, which confirms the association of Lactobacillus in healthy vaginal microbial communities. A more thoroughly comparison between BV-negative and BV-positive, including the retrieval of sequencing data from these samples, is necessary for getting more insight on BV influence on vaginal microbiota

Can a specific sub-group of biofilm- forming Gardnerella vaginalis strains be the real causative agent of bacterial vaginosis?

In the past half century, bacterial vaginosis (BV) has been a controversial topic in medical microbiology, and despite the wealth of information on this topic, the etiological agent has not yet been definitively identified [1]. The first advances on BV pointed Gardnerella vaginalis as the infectious causative agent of BV [2] but soon after it was found that G. vaginalis was also present in healthy women [3]. Additionally, G. vaginalis was not able to cause BV consistently. Furthermore, other microorganisms started to be associated with BV, and this resulted in a shift in the paradigm to that of a multispecies infection. However, epidemiological data revealed inconsistencies with this latter theory [4]. A couple of years ago the first descriptions of multispecies biofilm communities were described in BV [5]. Interestingly, G. vaginalis was present in most cases and accounted for the majority of the biofilm biomass. Further studies demonstrated that biofilm-forming G. vaginalis presented higher tolerance to external stresses [6]. Taking these data into consideration, we hypothesized that strains of G. vaginalis that were able to form biofilms could be the causative agent of BV. To test our hypothesis, we isolated more than 30 bacterial species from BV patients and also several strains of G. vaginalis from healthy women, and tested biofilm forming ability, initial adhesion to human vaginal cells, cytotoxicity activity, antimicrobial resistance and gene expression of know virulent genes. Our results revealed that G. vaginalis outcompeted all the other bacterial species in the initial adhesion to the epithelial cells. Furthermore, when comparing BV-associated G. vaginalis strains to strains isolated from healthy women, we found that all 7strains from BV were more virulent than the 7 strains colonizing healthy women, as measured by the higher cytotoxicity and the higher initial adhesion to epithelial cells. No significant differences were found in antimicrobial resistance profiles. Interestingly, no significant differences in expression of known virulence genes were detected, suggesting that the higher virulence of the BV-associated G. vaginalis was due to a yet unknown virulence determinant. We then tested virulent G. vaginalis against other known BV-associated anaerobe pathogens, namely Mobiluncus mulieris, Atopobium vaginae, Prevotella bivia and Fusobacteria nucleatum in mixed biofilm formation quantification. Interestingly, while the other tested anaerobes did not reveal a higher initial adhesion, they did enhance biofilm formation by G. vaginalis. Overall, our data suggests that virulent variants of G. vaginalis have the potential to be the etiological agent of BV, while acknowledging that other anaerobes do enhance G. vaginalis virulence

Functional analysis of virulence potential from Gardnerella vaginalis and other anaerobes commonly associated with Bacterial vaginosis

In the past half century, bacterial vaginosis (BV) has been a controversial topic in medical microbiology, and despite the wealth of information on this topic, the etiological agent has not yet been definitively identified [1]. The first advances on BV pointed Gardnerella vaginalis as the infectious causative agent of BV [2] but soon after it was found that G. vaginalis was also present in healthy women [3]. Additionally, G. vaginalis was not able to cause BV consistently. Furthermore, other microorganisms started to be associated with BV, and this resulted in a shift in the paradigm to that of a multispecies infection. However, epidemiological data revealed inconsistencies with this latter theory [4]. A couple of years ago the first descriptions of multispecies biofilm communities were described in BV [5]. Interestingly, G. vaginalis was present in most cases and accounted for the majority of the biofilm biomass. Further studies demonstrated that biofilm-forming G. vaginalis presented higher tolerance to external stresses [6]. Taking these data into consideration, we hypothesized that strains of G. vaginalis that were able to form biofilms could be the causative agent of BV. To test our hypothesis, we isolated more than 30 bacterial species from BV patients and also several strains of G. vaginalis from healthy women, and tested biofilm forming ability, initial adhesion to human vaginal cells, cytotoxicity activity, antimicrobial resistance and gene expression of know virulent genes. Our results revealed that G. vaginalis outcompeted all the other bacterial species in the initial adhesion to the epithelial cells. Furthermore, when comparing BV-associated G. vaginalis strains to strains isolated from healthy women, we found that all 7strains from BV were more virulent than the 7 strains colonizing healthy women, as measured by the higher cytotoxicity and the higher initial adhesion to epithelial cells. No significant differences were found in antimicrobial resistance profiles. Interestingly, no significant differences in expression of known virulence genes were detected, suggesting that the higher virulence of the BV-associated G. vaginalis was due to a yet unknown virulence determinant. We then tested virulent G. vaginalis against other known BV-associated anaerobe pathogens, namely Mobiluncus mulieris, Atopobium vaginae, Prevotella bivia and Fusobacteria nucleatum in mixed biofilm formation quantification. Interestingly, while the other tested anaerobes did not reveal a higher initial adhesion, they did enhance biofilm formation by G. vaginalis. Overall, our data suggests that virulent variants of G. vaginalis have the potential to be the etiological agent of BV, while acknowledging that other anaerobes do enhance G. vaginalis virulence