António Machado

Fluorescence in situ hybridization method using peptide nucleic acid probes for rapid detection of Lactobacillus and Gardnerella spp.

BackgroundBacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples.ResultsTherefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.ConclusionsThis quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

Interactions between Lactobacillus crispatus and bacterial vaginosis (BV)-associated bacterial species in initial attachment and biofilm formation.

Certain anaerobic bacterial species tend to predominate the vaginal flora during bacterial vaginosis (BV), with Gardnerella vaginalis being the most common. However, the exact role of G. vaginalis in BV has not yet been determined. The main goal of this study was to test the hypothesis that G. vaginalis is an early colonizer, paving the way for intermediate (e.g., Fusobacterium nucleatum) and late colonizers (e.g., Prevotella bivia). Theoretically, in order to function as an early colonizer, species would need to be able to adhere to vaginal epithelium, even in the presence of vaginal lactobacilli. Therefore, we quantified adherence of G. vaginalis and other BV-associated bacteria to an inert surface pre-coated with Lactobacillus crispatus using a new Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) methodology. We found that G. vaginalis had the greatest capacity to adhere in the presence of L. crispatus. Theoretically, an early colonizer would contribute to the adherence and/or growth of additional species, so we next quantified the effect of G. vaginalis biofilms on the adherence and growth of other BV-associated species by quantitative Polymerase Chain Reaction (qPCR) technique. Interestingly, G. vaginalis derived a growth benefit from the addition of a second species, regardless of the species. Conversely, G. vaginalis biofilms enhanced the growth of P. bivia, and to a minor extent of F. nucleatum. These results contribute to our understanding of BV biofilm formation and the progression of the disorder.

Influence of Biofilm Formation by Gardnerella vaginalis and Other Anaerobes on Bacterial Vaginosis

Bacterial vaginosis (BV) is the worldwide leading vaginal disorder among women of reproductive age. BV is characterized by the replacement of beneficial lactobacilli and the augmentation of anaerobic bacteria. Gardnerella vaginalis is a predominant bacterial species, but BV is also associated with other numerous anaerobes, such as Atopobium vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum, and Peptoniphilus species. Currently, the role of G. vaginalis in the etiology of BV remains a matter of controversy. However, it is known that, in patients with BV, a biofilm is usually formed on the vaginal epithelium and that G. vaginalis is typically the predominant species. So, the current paradigm is that the establishment of a biofilm plays a key role in the pathogenesis of BV. This review provides background on the influence of biofilm formation by G. vaginalis and other anaerobes, from the time of their initial adhesion until biofilm formation, in the polymicrobial etiology of BV and discusses the commensal and synergic interactions established between them to understand the phenotypic shift of G. vaginalis biofilm formation to BV establishment.

Reciprocal Interference between Lactobacillus spp. and Gardnerella vaginalis on Initial Adherence to Epithelial Cells

Bacterial vaginosis (BV) is the most common vaginal disorder in women of child-bearing age. It is widely accepted that the microbial switch from normal microflora to the flora commonly associated with BV is characterized by a decrease in vaginal colonization by specific Lactobacillus species together with an increase of G. vaginalis and other anaerobes. However, the order of events leading to the development of BV remains poorly characterized and it is unclear whether the decrease in lactobacilli is a cause or a consequence of the increase in the population density of anaerobes. Our goal was to characterize the interaction between two Gardnerella vaginalis strains, one of which was isolated from a healthy woman (strain 5-1) and the other from a woman diagnosed with BV (strain 101), and vaginal lactobacilli on the adherence to cervical epithelial cells. In order to simulate the transition from vaginal health to BV, the lactobacilli were cultured with the epithelial cells first, and then the G. vaginalis strain was introduced. We quantified the inhibition of G. vaginalis adherence by the lactobacilli and displacement of adherent lactobacilli by G. vaginalis. Our results confirmed that pathogenic G vaginalis 101 had a higher capacity for adhesion to the cervical epithelial cells than strain 5-1. Interestingly, strain 101 displaced L. crispatus but not L. iners whereas strain 5-1 had less of an effect and did not affect the two species differently. Furthermore, L. iners actually enhanced adhesion of strain 101 but not of strain 5-1. These results suggest that BV-causing G. vaginalis and L. iners do not interfere with one another, which may help to explain previous reports that women who are colonized with L. iners are more likely to develop BV.

Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples

Lactobacillus species constitute one of the dominant and beneficial bacteria in our body and are used in developed countries as a microbial adjuvant. Identification of these probiotic bacteria is traditionally performed by culture-based techniques. However, such methods are very time-consuming and can give inaccurate results, especially when Lactobacillus is present in mixed bacterial complex communities. Our study aimed to accurately identify Lactobacillus spp. using a novel Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) probe. The probe (Lac663) was tested on 36 strains belonging to different Lactobacillus species and on 20 strains of other bacterial species. The sensitivity and specificity of the method were 100% (95% confidence interval (CI), 88.0 to 100.0%) and 95.0% (95% CI, 73.1 to 99.7%), respectively. Additionally, we tested the applicability of the method on milk samples added with Lactobacillus strains at probiotic range concentrations and other taxonomically related bacteria, as well as pathogenic bacteria. The Lac663 probe bound exclusively to Lactobacillus strains and the described PNA-FISH method was capable of directly quantifying Lactobacillus spp. in concentrations at which these potential probiotic bacteria are considered to have an effective benefit on human health.

Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis.

Quantitative analysis of initial adhesion of bacterial vaginosis-associated anaerobes to ME-180 cells

Bacterial vaginosis is the leading vaginal disorder but the transition from health to this dysbiotic condition remains poorly characterized. Our goal was to quantify the ability of BV-associated anaerobes to adhere to epithelial cells in the presence of lactobacilli. Gardnerella vaginalis outcompeted Lactobacillus crispatus and Lactobacillus iners actually enhanced its adherence.

In silico vs in vitro analysis of primer specificity for the detection of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus spp.

BackgroundBacterial 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.FindingsHere 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.ConclusionsBy 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.

Multiplex Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) for diagnosis of bacterial vaginosis

Fluorescence in situ hybridization (FISH) is a molecular method used to identify and quantify microorganisms in a wide range of samples. This technique combines the simplicity of microscopic observation and the specificity of DNA/rRNA hybridization, allowing detection of selected bacterial species and morphologic visualization. Here, we describe a quantitative molecular diagnosis of bacterial vaginosis, based on the classical Nugent score. Our probes are able to differentiate Lactobacillus spp. and Gardnerella vaginalis from the other undefined bacterial species considered in the Nugent score.

Bacterial identification of the vaginal microbiota in Ecuadorian pregnant teenagers: an exploratory analysis

Background Bacterial vaginosis (BV) is a microbial imbalance (i.e., dysbiosis) that can produce serious medical effects in women at childbearing age. Little is known, however, about the incidence of BV or vaginal microbiota dysbiosis in pregnant teenagers in low and middle-income countries such as Ecuador. The scope of this exploratory analysis was to study the relationship between epidemiologic and microbial risk factors. Among the microbiology risk factors this study investigated five Lactobacillus species, two of them know in preview studies as microbiology risk factors for BV development (Lactobacillus acidophilus and Lactobacillus iners), and the last three known for being associated with a healthy vaginal tract (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii). In addition, fastidious anaerobes known to be microbial risk factors for BV development in pregnant teenagers were searched as well, more exactly, Gardnerella vaginalis, Atopobium vaginae and Mobiluncus mulieris. Methods Ninety-five healthy adolescent pregnant women, visiting a secondary level hospital in Quito, Ecuador, were enrolled into the study in 2015. The enrolled patients were between 10 to 13 weeks of pregnancy. Four epidemiological risk factors were collected in a survey: age, civil status, sexual partners and condom use. Also, vaginal pH was measured as a health risk factor. DNA was extracted from endocervical and exocervical epithelia from all the patients’ samples. PCR analysis was performed in order to characterize the presence of the eight bacterial species known as risk factors for BV development, targeting three anaerobes and five Lactobacillus species. Univariate and multivariate analysis were performed to identify associated factors for the presence of anaerobic species using logistic regression. Results The 95 vaginal microflora samples of these teenagers were analyzed. Two of the bacterial species known to cause BV: A. vaginae (100%) and G. vaginalis (93.7%) were found in high prevalence. Moreover, the most predominant bacterial Lactobacillus species found in the pregnant teenagers’ vaginal tract were L. crispatus (92.6%), L. iners (89.5%) and L. acidophilus (87.4%). In addition, the average vaginal pH measured in the study population was 5.2, and high pH was associated with the presence of the three-anaerobic species (p = 0.001). Finally, L. jensenii’s presence in the study decreased in 72% the occupation of the three anaerobes. Discussion This work identified a high pH as a risk factor for BV anaerobes’ presence in adolescent pregnant women. Moreover, this study identified L. crispatus, L. iners and L. acidophilus to be the most abundant species in our study population. From all fastidious anaerobes analyzed in this study, A. vaginae was present in all pregnant teenagers. To conclude, L. jensenii could be a potential healthy vaginal microbiota candidate in pregnant teenagers and should be further analyzed in future studies.