Selection of New Probiotics for Endometrial Health.

Microbiota is a crucial player in gynecologic health, in which bacteria can shift to a dysbiotic state triggering a pathogenic process. Based on an ecological understanding of the problem, the aim of this study is to select a potential probiotic strain to improve female reproductive tract based on its capacity to initially lower pH and to promote the reduction of pathogenic bacteria. Based on this rationale, strain Lactobacillus rhamnosus BPL005 was initially selected for its capacity to reduce in vitro pH levels and produce organic acids. Subsequently, strain L. rhamnosus BPL005 (CECT 8800) was demonstrated to have a protective role on endometrial infections in an in vitro model of bacterial colonization of primary endometrial epithelial cells with Atopobium vaginae, Gardnerella vaginalis, Propionibacterium acnes, and Streptococcus agalactiae. In this model, BPL005 when co-cultured with those pathogens was shown to lower pH and to produce organic acids, being lactic acid the most relevant. The co-cultivation of strain L. rhamnosus BPL005 with tested reference pathogens produced a significant reduction in P. acnes and St. agalactiae levels and a non-significant reduction in A. vaginae and G. vaginalis. The colonization of L. rhamnosus BPL005 in the culture decreased IL-6, IL-8, and MCP-1, heightened in the presence of pathogens, and increased IL-1RA and IL-1 beta. Finally, safety was evaluated showing no signs of cytotoxicity, irritation in vaginal tests, or allergic contact dermatitis potential through the Local Lymph Node Assay. Overall, these results show the potential of L. rhamnosus BPL005 strain as a probiotic in gynecological health.

Interaction of Gardnerella vaginalis and Vaginolysin with the Apical versus Basolateral Face of a Three-Dimensional Model of Vaginal Epithelium.

Studies have implicated Gardnerella vaginalis as an important etiological agent in bacterial vaginosis (BV). It produces a cholesterol-dependent cytolysin, vaginolysin (VLY). In this study, we sought to characterize the interaction between vaginal epithelium, G. vaginalis, and VLY using EpiVaginal tissues from MatTek. These tissues are three-dimensional and have distinct apical and basolateral sides, enabling comparison of the effects of G. vaginalis and VLY following exposure to either side. We measured cytotoxicity, cytokine production, and bacterial growth, following apical versus basolateral exposure. G. vaginalis exhibited more-rapid growth in coculture with the tissue model when it was exposed to the apical side. VLY permeabilized cells on the basolateral side of the tissues but failed to permeabilize apical epithelial cells. Cytokine secretion in response to VLY and G. vaginalis also depended on the polarity of exposure. VLY did not cause significant changes in cytokine levels when exposed apically. Apical tissue challenge by G. vaginalis appeared to dampen the inflammatory response, as decreases in granulocyte-macrophage colony-stimulating factor (GM-CSF) (6.6-fold), RANTES (14.8-fold), and interferon gamma inducible protein 10 kDa (IP-10) (53-fold) and an increase in interleukin-1 receptor antagonist (IL-1ra) (5-fold) were observed. In vivo, G. vaginalis normally colonizes the apical face of the vaginal epithelium. Results from this study suggest that while G. vaginalis may grow on the apical face of the vaginal epithelium, its VLY toxin does not target these cells in this model. This phenomenon could have important implications regarding colonization of the vagina by G. vaginalis and may suggest an explanation for the lack of an overt immune response to this organism.

Anti-inflammatory effect of two Lactobacillus strains during infection with Gardnerella vaginalis and Candida albicans in a HeLa cell culture model.

Lactobacilli are the dominant bacteria of the vaginal tract of healthy women and they play a major role in the maintenance of mucosal homeostasis, preventing genital infections, such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). It is now known that one mechanism of this protection is the influence that lactobacilli can exert on host immune responses. In this context, we evaluated two Lactobacillus strains (L. plantarum 59 and L. fermentum 137) for their immunomodulatory properties in response to Gardnerella vaginalis (BV) or Candida albicans (VVC) infections in a HeLa cell infection model. G. vaginalis and C. albicans triggered the secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-8) and the activation of NF-κB in HeLa cells, in contrast to L. plantarum 59 and L. fermentum 137. Treatments with the Lactobacillus strains or their cell-free supernatants before (pre-treatment) or after (post-treatment) the challenge with the pathogens resulted in decreased secretion of pro-inflammatory cytokines and decreased activation of NF-κB. The treatments with Lactobacillus strains not only decreased the secretion of IL-8, but also its expression, as confirmed by gene reporter luciferase assay, suggesting transcription-level control by lactobacilli. In conclusion, L. plantarum 59 and L. fermentum 137 were confirmed to have an anti-inflammatory effect against G. vaginalis and C. albicans and they were able to influence signalling in NF-κB pathway, making them interesting candidates as probiotics for the prevention or treatment of BV and VVC.

Poly(ethylene glycol) (PEG)-lactic acid nanocarrier-based degradable hydrogels for restoring the vaginal microenvironment.

Women with bacterial vaginosis (BV) display reduced vaginal acidity, which make them susceptible to associated infections such as HIV. In the current study, poly(ethylene glycol) (PEG) nanocarrier-based degradable hydrogels were developed for the controlled release of lactic acid in the vagina of BV-infected women. PEG-lactic acid (PEG-LA) nanocarriers were prepared by covalently attaching lactic acid to 8-arm PEG-SH via cleavable thioester bonds. PEG-LA nanocarriers with 4 copies of lactic acid per molecule provided controlled release of lactic acid with a maximum release of 23% and 47% bound lactic acid in phosphate buffered saline (PBS, pH7.4) and acetate buffer (AB, pH4.3), respectively. The PEG nanocarrier-based hydrogels were formed by cross-linking the PEG-LA nanocarriers with 4-arm PEG-NHS via degradable thioester bonds. The nanocarrier-based hydrogels formed within 20 min under ambient conditions and exhibited an elastic modulus that was 100-fold higher than the viscous modulus. The nanocarrier-based degradable hydrogels provided controlled release of lactic acid for several hours; however, a maximum release of only 10%-14% bound lactic acid was observed possibly due to steric hindrance of the polymer chains in the cross-linked hydrogel. In contrast, hydrogels with passively entrapped lactic acid showed burst release with complete release within 30 min. Lactic acid showed antimicrobial activity against the primary BV pathogen Gardnerella vaginalis with a minimum inhibitory concentration (MIC) of 3.6 mg/ml. In addition, the hydrogels with passively entrapped lactic acid showed retained antimicrobial activity with complete inhibition G. vaginalis growth within 48 h. The results of the current study collectively demonstrate the potential of PEG nanocarrier-based hydrogels for vaginal administration of lactic acid for preventing and treating BV.

Vaginolysin drives epithelial ultrastructural responses to Gardnerella vaginalis.

Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-ß-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.

Lactobacillus johnsonii HY7042 ameliorates Gardnerella vaginalis-induced vaginosis by killing Gardnerella vaginalis and inhibiting NF-κB activation.

Hydrogen peroxide-producing lactic acid bacteria (LAB) were isolated from women's vaginas and their anti-inflammatory effects against Gardnerella vaginalis-induced vaginosis were examined in ß-estradiol-immunosuppressed mice. Oral and intravaginal treatment with five LABs significantly decreased viable G. vaginalis numbers in vaginal cavities and myeloperoxidase activity in mouse vaginal tissues. Of the LABs examined, Lactobacillus johnsonii HY7042 (LJ) most potently inhibited G. vaginalis-induced vaginosis. This LAB also inhibited the expressions of IL-1ß, IL-6, TNF-α, COX-2, and iNOS, and the activation of NF-κB in vaginal tissues, but increased IL-10 expression. Orally administered LJ (0.2×10(8) CFU/mouse) also inhibited the expression of TNF-α by 91.7% in ß-estradiol-immunosuppressed mice intraperitoneally injected with LPS. However, it increased IL-10 expression by 63.3% in these mice. Furthermore, LJ inhibited the expressions of the pro-inflammatory cytokines, TNF-α and IL-1ß, and the activation of NF-κB in lipopolysaccharide-stimulated peritoneal macrophages. LJ also killed G. vaginalis attached with and without HeLa cells. These findings suggest that LJ inhibits bacterial vaginosis by inhibiting the expressions of COX-2, iNOS, IL-1ß, and TNF-α by regulating NF-κB activation and by killing G. vaginalis, and that LJ could ameliorate bacterial vaginosis.

Correlation of Trichomonas vaginalis to bacterial vaginosis: a laboratory-based study.

BACKGROUND: This study aimed to define the occurrence of different organisms causing vulvovaginitis; to evaluate different laboratory methods used for diagnosis of Trichomonas vaginalis (T. vaginalis); and to evaluate the direct score system and clue cell method compared with culture for diagnosis of bacterial and T. vaginalis vaginosis. METHODOLOGY: Clinical and laboratory evaluations were performed for 110 patients. Laboratory methods used for bacteriological diagnosis were direct Gram staining for clue cells and scoring by Nugent score system and bacterial culture. T. vaginalis was identified by wet mount microscopic examination, culture, direct Gram, Giemsa staining and acridine orange (AO). RESULTS: The Nugent score method revealed that the sensitivity and specificity for diagnosis of vaginal discharge by direct rapid microscopic methods were 30% and 80% and for clue cells sensitivity and specificity were 37% and 75% respectively for diagnosis of bacterial vaginosis compared to culture. For diagnosis of T. vaginalis, the Nugent score method revealed that the sensitivity and specificity were 60% and 90% respectively, and for clue cells 75% and 80% respectively. For microcopic methods used for T. vaginalis only, the Gram stain and Giemsa stain sensitivities were poor (15.2% and 48.5%, respectively). Wet mount showed reasonable sensitivity of 75.8%. Acridine orange sensitivity was 93.9% and specificity was 97.5%, CONCLUSION: Prevalent pathogens associated with vaginitis were (Gardnerella vaginalis) G. vaginalis, T. vaginalis and Mycoplasma hominis (M. hominis). Wet mount microscopic examination, acridine orange, and high Nugent score were found as rapid and sensitive methods for diagnosis of T. vaginalis.

Improving Gram-stained reproducible result by further adding clue cells in diagnosing bacterial vaginosis.

The reproducibility of interpretation in diagnosing bacterial vaginosis may be enhanced by adding pus cells and clue cells into two different criteria, developed by Spiegel et al. and Nugent et al. The purpose of study was designed to find out which parameter was more reproducible. 100 patients were collected with the diagnosis of bacterial vaginosis as an experimental group, while the other 100 patients who were with routine Papanicolaou smears in gynecologic clinic the collected as a control group. Two slides, including the original and reproducible ones, were obtained from vaginal smears for each patient. Three technicians read the slides randomly by using two different criteria, plus pus cells and clue cells. This showed the agreement for clue cells is the best method regardless of experimental group or control group (Kappa values between 0.708 and 1.000). The intra-observer agreement for the diagnosis of bacterial vaginosis by the method of Nugent et al. is superior to the method of Spiegel et al. Our data show the comparison of Amsel criteria versus Nugent criteria for the diagnosis of bacterial vaginosis with sensitivity of 88.9%, specificity of 55.4%, negative positive value of 62.1%, and positive predictive value of 85.8%. Moreover, our data also demonstrate the comparison of Amsel criteria versus the diagnosis either based on Nugent criteria or the presence of clue cells with sensitivity of 95.7%, specificity of 56.7%, negative positive value of 81.2%, and positive predictive value of 87.1%. The results demonstrate further adding score of the clue cells can enhance the reproducible diagnosis of bacterial vaginosis, which is superior to the methods of Nugent et al. and Spiegel et al.

Evidence for a commensal, symbiotic relationship between Gardnerella vaginalis and Prevotella bivia involving ammonia: potential significance for bacterial vaginosis.

Six strains of Prevotella bivia and 4 of Gardnerella vaginalis were examined for nutrient substrate utilization as part of ongoing studies on the pathogenesis of bacterial vaginosis. Addition of single amino acids to vaginal defined medium (VDM) was stimulatory to the growth of P. bivia but not to G. vaginalis. However, peptides significantly promoted the growth of both organisms. Growth of P. bivia in VDM and VDM supplemented with either amino acids or peptone was accompanied by net ammonia production, while growth of G. vaginalis under the same conditions resulted in net ammonia utilization. Ammonia-enriched supernatants from the growth of P. bivia in peptone-supplemented VDM were stimulatory to G. vaginalis growth. However, ammonia-reduced supernatants from G. vaginalis growth in peptone-supplemented VDM had a neutral effect on P. bivia growth. A commensal relationship between P. bivia to G. vaginalis is proposed, with ammonia flow as a mechanism to support this hypothesis.

Clue cells in bacterial vaginosis: immunofluorescent identification of the adherent gram-negative bacteria as Gardnerella vaginalis.

Clue cells are epithelial cells covered by adherent gram-negative rods, observed in vaginal smears from women with bacterial vaginosis. Immunofluorescence studies were used to identify the gram-negative bacteria adhering to clue cells. Specific antisera to four common gram-negative vaginal bacteria (Gardnerella, Bacteroides, Fusobacterium, and Mobiluncus) were prepared by long-term, multiple, small-inoculum immunization of rabbits. Cross-reactivity with heterologous common vaginal bacteria was removed by absorption against whole cells of heterologous bacteria and by serial dilution. Gardnerella vaginalis was most often observed adhering to the surface of clue cells and was detected on the surface of exfoliated vaginal epithelial cells significantly more frequently and in higher numbers than were Mobiluncus, Bacteroides, and Fusobacterium, suggesting that this species of gram-negative bacteria is responsible for clue cell formation.

Gardnerella vaginalis is associated with other sexually transmittable microorganisms in the male urethra.

In a prospective study, urethral swabs were taken from 544 men presented to an STD clinic, 118 with and 426 without urethritis, and examined by microscope and/or culture for G. vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, Ureaplasma urealyticum, Mycoplasma hominis, Candida species and Trichomonas vaginalis. G. vaginalis was isolated from 4.2% of the males with urethritis and from 6.3% of those without urethritis. Using loglinear analysis, the following associations were significant (p less than 0.05): three-way: G. vaginalis, U. urealyticum, C. trachomatis; two-way: G. vaginalis, U. urealyticum and G. vaginalis, M. hominis and U. urealyticum, M. hominis. It is concluded that G. vaginalis is associated with genital mycoplasmas not only in the female, but also in the male urogenital tract.

The prevalence, six-month persistence, and predictive values of laboratory indicators of bacterial vaginosis (nonspecific vaginitis) in asymptomatic women.

The natural course of signs and laboratory test findings indicative of bacterial vaginosis was followed in an observational noninterventive 6-month longitudinal study of 270 asymptomatic women. Only the minority of positive Gardnerella vaginalis cultures (5 of 33), wet mount clue cells (5 of 14), sniff tests (3 of 11), Papanicolaou smear clue cells (0 of 5), and discharge consistent with bacterial vaginosis (11 of 49) persisted in the absence of therapy. While these four laboratory parameters as well as chromatographic succinate/lactate ratios (performed only on the final visit) were abnormal significantly more often in patients with abnormal discharge than in those with normal discharge (p = 0.006, p less than 0.0001, p less than 0.0001, p = 0.0003, and p = 0.002, respectively), all were insensitive predictors of abnormal discharge with sensitivities ranging between 10.6% and 20.2% and abnormal test predictive values between 30.6% and 65.2%. We conclude that G. vaginalis represents indigenous flora in some normal women and that therapy is unwarranted for the incidental finding of a positive laboratory indicator of bacterial vaginosis in the patient without symptoms.

Characterization of Gardnerella vaginalis by gas chromatography.

The principal objective of this study was the characterization of Gardnerella vaginalis by gas chromatography. Thirty-eight isolates and the type strain, ATCC 14018, of G. vaginalis were studied. Hexadecanoic (16:0), octadecenoic (18:1) and octadecanoic (18:0) were the major fatty acids detected. Only insignificant differences between the various isolates could be found. The gas chromatographic analysis of G. vaginalis revealed a characteristic pattern. Gas chromatography in combination with selective growth conditions provides a method for rapid and exact identification.

Comparison of microscopic and cultural findings in the diagnosis of Gardnerella vaginalis infection.

The diagnosis of Gardnerella vaginalis infection on the basis of microscopic and cultural findings was compared. A total of 340 specimens of vaginal secretion were Gram stained and plated on a medium selective for Gardnerella vaginalis. Positive culture was obtained in 165 cases. Microscopy was unequivocally positive in 95, doubtful in 58 and negative in 187. Positive microscopy was confirmed by culture in 99%. On the other hand, 21% of the negative microscopy results gave a false negative diagnosis. Specimens for which microscopy was doubtful were culture positive in 53% of the cases, including 12% with heavy growth. Thus, positive microscopy proved to be sufficient for a reliable diagnosis of Gardnerella vaginalis infection. However, in specimens with negative or doubtful microscopic findings, additional culture is recommended.