Crystal Violet Staining Alone Is Not Adequate to Assess Synergism or Antagonism in Multi-Species Biofilms of Bacteria Associated With Bacterial Vaginosis

Bacterial Vaginosis (BV) involves the presence of a multi-species biofilm adhered to vaginal epithelial cells, but its in-depth study has been limited due to the complexity of the bacterial community, which makes the design of in vitro models challenging. Perhaps the most common experimental technique to quantify biofilms is the crystal violet (CV) staining method. Despite its widespread utilization, the CV method is not without flaws. While biofilm CV quantification within the same strain in different conditions is normally accepted, assessing multi-species biofilms formation by CV staining might provide significant bias. For BV research, determining possible synergism or antagonism between species is a fundamental step for assessing the roles of individual species in BV development. Herein, we provide our perspective on how CV fails to properly quantify an in vitro triple-species biofilm composed of Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, and Prevotella bivia, three common BV-associated bacteria thought to play key roles in incident BV pathogenesis. We compared the CV method with total colony forming units (CFU) and fluorescence microscopy cell count methods. Not surprisingly, when comparing single-species biofilms, the relationship between biofilm biomass, total number of cells, and total cultivable cells was very different between each tested method, and also varied with the time of incubation. Thus, despite its wide utilization for single-species biofilm quantification, the CV method should not be considered for accurate quantification of multi-species biofilms in BV pathogenesis research.

The effect of vaginal probiotic therapy on the outcome of papillomavirus infection

Aim. To study the effect of vaginal probiotic therapy on the outcome of human papillomavirus (HPV) infection.Materials and methods. The study included HPV-infected patients: 29 patients with normal vaginal flora and 146 patients with a deficiency of vaginal lactobacilli, of which 117 patients received vaginal probiotic therapy. In samples obtained before and after the therapy, the effect of the probiotic on the change in the ratio of living, apoptotic, and necrotic vaginal epithelial cells after preliminary exposure to oxidative stress was studied.Results. It was found that probiotics reduce the number of infected epithelial cells that survived the oxidative damage and shift the balance of cell death forms towards apoptosis. Vaginal probiotic therapy in patients with a deficiency of lactobacilli increased the frequency of HPV elimination by 2.5 times and reduced the likelihood of treatment failure from 1.5 to 4 times, depending on the viral load. The probiotic therapy made the structure of HPV outcomes in Lactobacillus-deficient patients similar to that in patients with normal vaginal flora.Conclusion. Vaginal probiotic therapy improves outcomes of HPV infection in patients with a deficiency of lactobacilli by reducing the number of survived infected cells and shifting the cell death pattern towards apoptosis.

Characterization of a novel fructosyltransferase InuCA from Lactobacillus crispatus that attaches to the cell surface by electrostatic interaction

Fructosyltransferases (FTases), a group of carbohydrate-active enzymes, synthesize fructooligosaccharides (FOS) and fructans, which are promising prebiotics for human health. Here we originally identified a novel FTase InuCA from L. crispatus , a dominant species in the vaginal microbiotas of human. InuCA was characterized by a shortest C-terminus and the highest isoelectric point among the reported Lactobacillus FTases. InuCA was an inulosucrase and produced a serial of FOS using sucrose as substrate at a moderate temperature. Surprisingly, the C-terminal deletion mutant synthesized oligosaccharides with fructosyl chain longer than that of the wild type, suggesting that the C-terminal part blocked the binding of long-chain receptor. Moreover, InuCA bound to the cell surface by electrostatic interaction, which was dependent on the environmental pH and represented a distinctive binding mode in FTases. The catalytic and structural properties of InuCA will be contributed to the FTases engineering and the knowledge of the adaptation of L. crispatus in the vaginal environment. Importance L. crispatus is one of the most important species in human vaginal microbiotas and its persistence is strongly negatively correlated with the vaginal diseases. Our research reveals that a novel inulosucrase InuCA is present in L. cirspatus . InuCA keeps the ability to synthesize prebiotic fructo-oligosaccharides, although it lacks a large part of the C-terminal region compared to other FTases. Remarkably, the short C-terminus of InuCA blocks the transfructosylation activity for producing oligosaccharides with longer chain, which is meaningful to the directional modification of FTases and the oligosaccharide products. Besides the catalytic activity, InuCA is anchored on the cell surface dependent on the environmental pH and may be also involved in the adhesion of L. crispatus to the vaginal epithelial cells. Since L. crispatus plays an essential role in the normal vaginal micro-ecosystem, the described work will be helpful to elucidate the functional genes and colonization mechanism of the dominant species.

Impact of vaginal douching products on vaginal Lactobacillus, Escherichia coli and epithelial immune responses

We compared the effect of commercial vaginal douching products on Lactobacillus crispatus, L. jensenii, L. gasseri, L. iners, E. coli, and immortalized vaginal epithelial cells (VK2). All studied douching products (vinegar, iodine and baking soda based) induced epithelial cell death, and all inhibited growth of E. coli. Co-culture of vaginal epithelial cells with any of the lactobacilli immediately following exposure to douching products resulted in a trend to less human cell death. However, co-culture of epithelial cells with L. iners was associated with higher production of IL6 and IL8, and lower IL1RA regardless of presence or type of douching solution. Co-culture with L. crispatus or L. jensenii decreased IL6 production in the absence of douches, but increased IL6 production after exposure to vinegar. Douching products may be associated with epithelial disruption and inflammation, and may reduce the anti-inflammatory effects of beneficial lactobacilli.

pH Stress Mediated Alteration in Protein Composition and Reduction in Cytotoxic Potential of Gardnerella vaginalis Membrane Vesicles

The vagina of healthy women is predominantly colonized by lactobacilli but it also harbors a limited proportion of certain anaerobes such as Gardnerella vaginalis. An increase in G. vaginalis along with other anaerobes on account of perturbation in the vaginal microbiota is associated with bacterial vaginosis (BV). Although strategies adopted by G. vaginalis for survival and pathogenesis in a conducive environment (i.e., high vaginal pH, characteristic of BV) have been previously studied, the approaches potentially employed for adaptation to the low pH of the healthy vagina are unknown. In the present study, we investigated the effect of acidic stress on the modulation of the production and function of membrane vesicles (MVs) of G. vaginalis. pH stress led to a distortion of the bacterial cell morphology as well as an altered biogenesis of MVs, as revealed by transmission electron microscopy (TEM). Both qualitative and quantitative differences in protein content of MVs produced in response to pH stress were observed by flow cytometry. A significant change in the protein composition characterized by presence of chaperones despite a reduction in number of proteins was also noted in the stress induced MVs. Further, these changes were also reflected in the reduced cytotoxic potential toward vaginal epithelial cells. Although, these findings need to be validated in the in vivo settings, the modulation of G. vaginalis MV biogenesis, composition and function appears to reflect the exposure to acidic conditions prevailing in the host vaginal mileu in the absence of vaginal infection.

Human milk oligosaccharides reduce murine group B Streptococcus vaginal colonization with minimal impact on the vaginal microbiota

Group B Streptococcus (GBS) colonizes the vaginal mucosa of a significant percentage of healthy women and is a leading cause of neonatal bacterial infections. Currently, pregnant women are screened in the last month of pregnancy and GBS-positive women are given antibiotics during parturition to prevent bacterial transmission to the neonate. Recently, human milk oligosaccharides (HMOs) isolated from breastmilk were found to inhibit GBS growth and biofilm formation in vitro, and women that make certain HMOs are less likely to be vaginally colonized with GBS. Using in vitro human vaginal epithelial cells and a murine vaginal colonization model, we tested the impact of HMO treatment on GBS burdens and the composition of the endogenous microbiota by 16S rRNA amplicon sequencing. HMO treatment reduced GBS vaginal burdens in vivo with minimal alterations to the vaginal microbiota. HMOs displayed potent inhibitory activity against GBS in vitro, but HMO pretreatment did not alter adherence of GBS or the probiotic Lactobacillus rhamnosus to human vaginal epithelial cells. Additionally, disruption of a putative GBS glycosyltransferase (Δsan_0913) rendered the bacterium largely resistant to HMO inhibition in vitro and in vivo but did not compromise its adherence, colonization, or biofilm formation in the absence of HMOs. We conclude that HMOs are a promising therapeutic bioactive to limit GBS vaginal colonization with minimal impacts on the vaginal microenvironment.

Medroxyprogesterone Acetate (MPA) Enhances HIV-1 Accumulation and Release in Primary Cervical Epithelial Cells by Inhibiting Lysosomal Activity

Medroxyprogesterone acetate (MPA) is one of the most widely used contraceptives in the world. Epidemiologic studies have uncovered a possible link between the use of MPA and an increased risk of HIV-1 transmission. However, the understanding of the mechanism is still limited. Our previous publication demonstrated that the lysosomal activity in human vaginal epithelial cells attenuated the trafficking of viral particles during HIV-1 transcytosis. In this study, we show that treating human primary cervical epithelial cells with MPA led to a reduction in lysosomal activity. This reduction caused an increase in the intracellular HIV-1 accumulation and, consequently, an increase in viral release. Our study uncovers a novel mechanism by which MPA enhances HIV-1 release in primary cervical epithelial cells, thus providing vital information for HIV intervention and prevention.

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

The role of 17β-estrogen in Escherichia coli adhesion on human vaginal epithelial cells via FAK phosphorylation

Estrogen, the predominant sex hormone, has been found to be related to the occurrence of vaginal infectious diseases. However, its role in the occurrence and development of bacterial vaginitis caused by Escherichia coli is still unclear. The objective of this study was to investigate the role of 17β-estrogen in E. coli adhesion on human vaginal epithelial cells. The vaginal epithelial cell line, VK2/E6E7, was used to study the molecular events induced by estrogen between E. coli and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation with cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by Western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα-FAK signaling cascade. The results showed that, following the stimulation with 1000 nM estrogen for 48 h, a transient activation of ERα and FAK was observed, as well as the increased average number of E. coli adhering to vaginal epithelial cell. In addition, estrogen-induced activation of ERa and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 μM concentration and acted for 1 h, and a decrease in the number of adherent E. coli was observed simultaneously. However, this inhibitory effect diminished as the concentration of estrogen increased. In conclusion, FAK and ERα signaling cascades were assosiated with the increasing E. coli adherence to vaginal epithelial cells, which was promoted by a certain concentration of estrogen.