Underwater plasma discharge temporally produces several reactive radicals and/or free chlorine molecules in water, which is responsible for antimicrobial activity. Hence, it can simply sanitize tap water without disinfectant treatment. Additionally, the spraying technique using cleaning water exploits deep application in the narrow and curved vaginal tract of patients. Herein, we attempted a clinical trial to evaluate the vaginal cleaning effect of spraying plasma-activated water (PAW) to patients with vaginitis (46 patients). The efficacy was compared with treatment with betadine antiseptics used to treat bacterial vaginosis (40 patients). To evaluate the cleaning effect, Gram staining of the vaginal secretions was conducted before and after spraying PAW or betadine treatment (BT). Consequently, PAW-sprayed (PAWS) patients (22.3%) showed a better vaginal cleaning effect against Gram-positive and -negative bacteria than BT patients (14.4%). Moreover, 18 patients in the BT group showed worsened vaginal contamination, whereas five patients in the PAWS group showed worsened vaginal contamination. Taken together, the noncontact method of spraying cleaning water to the vagina exhibited a reliable vaginal cleaning effect without further bacterial infection compared with BT. Therefore, we suggest a clinical application of the spraying method using PAW for vaginal cleaning to patients with vaginitis without disinfectants and antibiotics.

Vulvovaginal candidiasis (VVC) is characterized by symptomatic inflammatory responses in the vagina caused by Candida albicans and non-albicans Candida (NAC) species. The epidermal growth factor receptor (EGFR) -mitogen-activated protein kinase (MAPK) signaling pathway has been linked to immune responses of oral mucosa after C. albicans exposure, but whether this pathway plays a similar response in vaginal epithelial cells is not known. Here, we observed that phosphorylation of EGFR and p38 was continuously activated in vaginal epithelial cells by C. albicans strain SC5314. This differs markedly from oral epithelial cells, which respond in a biphasic manner in order to properly discriminate the morphology of C. albicans. When compared with SC5314, a highly azole-resistant C. albicans isolate 1052 can induce a stronger phosphorylated signal of EGFR and p38, while clinically-isolated NAC strains including C. tropicalis, C. glabrata, C. parapsilosis and C. auris trigger higher levels of phosphorylated ERK1/2 and c-Fos than C. albicans. Inhibition of EGFR significantly reduces inflammatory response and epithelial damage induced by C. albicans both in vitro and in vivo, while inhibition of p38 leads to significant repair of epithelial damage triggered by both C. albicans and NAC species. These results confirm the importance of the EGFR-MAPK signaling in VVC pathogenesis and highlight the remarkable immunogenic differences between C. albicans and NAC species in host-microbe interactions.

Clue cells (epithelial cells heavily covered with adherent bacteria) are an accepted clue to the diagnosis of bacterial vaginosis. However, the exact morphologic criteria of clue cells and bacterial adherence were never elaborated.

Gardnerella vaginalis (GV) is an anaerobic bacterial species involved in the pathogenesis of bacterial vaginosis (BV), a condition of vaginal dysbiosis associated with adverse pregnancy outcomes. GV strains are categorized into four clades, characterized by a different ability to produce virulence factors, such as sialidase. We investigated the distribution of GV clades and sialidase genes in the vaginal ecosystem of a cohort of pregnant women, assessing the correlations between GV clades and the whole vaginal microbiome. A total of 61 Caucasian pregnant women were enrolled. Their vaginal swabs, collected both at the first and third trimester of pregnancy, were used for (i) evaluation of the vaginal status by Nugent score, (ii) vaginal microbiome profiling by 16S rRNA sequencing, (iii) detection and quantification of GV clades and sialidase A gene by qPCR assays. DNA of at least one GV clade was detected in most vaginal swabs, with clade 4 being the most common one. GV clade 2, together with the presence of multiple clades (>2 simultaneously), were significantly associated with a BV condition. Significantly higher GV loads and sialidase gene levels were found in BV cases, compared to the healthy status. Clade 2 was related to the major shifts in the vaginal microbial composition, with a decrease in Lactobacillus and an increase in several BV-related taxa. As the number of GV clades detected simultaneously increased, a group of BV-associated bacteria tended to increase as well, while Bifidobacterium tended to decrease. A negative correlation between sialidase gene levels and Lactobacillus, and a positive correlation with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia were observed. Our results added knowledge about the interactions of GV clades with the inhabitants of the vaginal microbiome, possibly helping to predict the severity of BV and opening new perspectives for the prevention of pregnancy-related complications.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

Vulvovaginal candidiasis (VVC) is one of the most common types of vaginal infections in women around the world and is often underestimated by both patients and doctors. Research on the pathogenesis of fungal vaginal infections over the last 20 years has resulted in a closer understanding of the virulence factors involved in Candida epithelial invasion and their mechanisms of action. Recently, attention was drawn to the enormous complexity of the interaction between yeast-like fungi and host cells, as well as the level of complexity of the host's response to infection and their impact on the course and treatment of VVC. Our work provides a broad description of already known and some new reports on Candida virulence factors (such as phenotypic switching or biofilm formation capacity) and their importance for tissue invasion in VVC. At the same time, we also focus on interactions with host cells and local innate immune mechanisms involved in the response to vaginal fungal invasion that are now considered equally important in this case. The presented review describes the most important aspects of the still unknown pathogenicity of Candida associated with vaginal infections.

A deep comprehension of the vaginal ecosystem may hold promise for unraveling the pathophysiology of pregnancy and may provide novel biomarkers to identify subjects at risk of maternal-fetal complications. In this prospective study, we assessed the characteristics of the vaginal environment in a cohort of pregnant women throughout their different gestational ages and puerperium. Both the vaginal bacterial composition and the vaginal metabolic profiles were analyzed. A total of 63 Caucasian women with a successful pregnancy and 9 subjects who had a first trimester miscarriage were enrolled. For the study, obstetric examinations were scheduled along the three trimester phases (9-13, 20-24, 32-34 gestation weeks) and puerperium (40-55 days after delivery). Two vaginal swabs were collected at each time point, to assess the vaginal microbiome profiling (by Nugent score and 16S rRNA gene sequencing) and the vaginal metabolic composition (1H-NMR spectroscopy). During pregnancy, the vaginal microbiome underwent marked changes, with a significant decrease in overall diversity, and increased stability. Over time, we found a significant increase of Lactobacillus and a decrease of several genera related to bacterial vaginosis (BV), such as Prevotella, Atopobium and Sneathia. It is worth noting that the levels of Bifidobacterium spp. tended to decrease at the end of pregnancy. At the puerperium, a significantly lower content of Lactobacillus and higher levels of Gardnerella, Prevotella, Atopobium, and Streptococcus were observed. Women receiving an intrapartum antibiotic prophylaxis for Group B Streptococcus (GBS) were characterized by a vaginal abundance of Prevotella compared to untreated women. Analysis of bacterial relative abundances highlighted an increased abundance of Fusobacterium in women suffering a first trimester abortion, at all taxonomic levels. Lactobacillus abundance was strongly correlated with higher levels of lactate, sarcosine, and many amino acids (i.e., isoleucine, leucine, phenylalanine, valine, threonine, tryptophan). Conversely, BV-associated genera, such as Gardnerella, Atopobium, and Sneathia, were related to amines (e.g., putrescine, methylamine), formate, acetate, alcohols, and short-chain fatty-acids (i.e., butyrate, propionate).

The vaginal microbiota plays vital protection in women. This probiotic activity is caused not only by individual Lactobacillus species but also by its multi-microbial interaction. However, the probiotic activity promoted by multi-microbial consortia is still unknown. The aim of this study was the individual and collective analysis on the prevalence of five vaginal lactobacilli (Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus acidophilus) among healthy women and women with bacterial vaginosis (BV) or aerobic vaginitis (AV). PCR assays were realized on 436 vaginal samples from a previous study. Chi-square, univariable, and multivariable logistic regression analyses with the Benjamini-Hochberg adjustment evaluated associations between these lactobacilli and vaginal microbiota. Multi-microbial clustering model was also realized through Ward's Minimum Variance Clustering Method with Euclidean squared distance for hierarchical clustering to determine the probiotic relationship between lactobacilli and vaginal dysbiosis. Concerning the individual effect, L. acidophilus, L. jensenii, and L. crispatus showed the highest normalized importance values against vaginal dysbiosis (100%, 79.3%, and 74.8%, respectively). However, only L. acidophilus and L. jensenii exhibited statistical values (p = 0.035 and p = 0.050, respectively). L. acidophilus showed a significant prevalence on healthy microbiota against both dysbioses (BV, p = 0.041; and AV, p = 0.045). L. jensenii only demonstrated significant protection against AV (p = 0.012). Finally, our results evidenced a strong multi-microbial consortium by L. iners, L. jensenii, L. gasseri, and L. acidophilus against AV (p = 0.020) and BV (p = 0.009), lacking protection in the absence of L. gasseri and L. acidophilus.

Vaginosis is a condition experienced by most women at least once in their lifetime. This condition arises due to the imbalance in the microbiome of the vaginal ecosystem. Most of the pathogens of this disease are organisms which are commonly found in a normal healthy vagina. The vaginal microbiome is important as they act as a primary defence against secondary infections and Sexually transmitted diseases and infections (STDs and STIs). The vagina is mostly dominated by Lactobacillus along with other microbes including Gardnerella vaginalis, Atopobium vaginae., Prevotella spp., Mobiluncus spp., etc. Vaginal microbiome also includes Candida albicans and other species of the genus. The ratio in which these species are present varies from person to person and the dominant species decides the whether a vagina is "normal" or not. Lactobacillus dominated vagina is considered normal and if dominated by Gardnerella and such it is considered to be Bacterial vaginosis (BV) and similarly for Vulvovaginal Candidiasis (VVC). The microbiome also undergoes changes during menstrual cycles and menopausal stages. Due to the dynamic nature of this microbiome, it is tough to perfectly restore the balance. But several treatments are currently available with antibiotics like Clindamycin and derivatives of 5-nitroimidazole drugs like Metronidazole. The extensive use and the non-adherence to the treatment regimen has led to drug resistance through biofilm formation, efflux pumps, single nucleotide polymorphisms and resulting recurrent episode of vaginosis in women. Alternative medicines, preparations from plant sources, anti-microbial peptides and nano formulations are also being explored. Most of these medicines tend to focus on reducing the pathogen load rather than restoring the balance of the ecosystem. Vaginal microbiome transplant, an effort to restore the normalcy in the vaginal environment is becoming a popular treatment. In this review we discuss about the types of vaginosis, available treatments, challenges in treating the condition and the new drugs that are under investigation.

Vulvar candidiasis (VVC) is a vaginitis caused by vaginal mucosa infection of Candida, which greatly impairs women's health. Although there are more and more thiazoles on the market, new classes of antifungal drugs are still missing, it is still challenging to treat azole-resistant candidal vaginitis. We found that L-Se-methylselenocysteine (L-SeMC) could effectively inhibit the growth of Candida albicans, reduce the density and length of the mycelia. To extend the retention time of L-SeMC in the vaginal tract and enhance its therapeutic effect for VVC, a mucoadhesive thermogel (NAC-HA thermogel) was successfully synthesized and prepared. The gelation window was around 29-56 °C for L-SeMC loaded mucoadhesive thermogel (L-SeMC@NAC-HA thermogel), which exhibited a sustained release profile in the in vitro release study and an extended retention time in the vaginal tract. Besides, L-SeMC@NAC-HA thermogel exhibited a good safety profile in the in vivo safety study. The in vivo anti-VVC effect was examined in a rat VVC model and L-SeMC@NAC-HA thermogel significantly reduced the number of Candida albicans in the vaginal secreta, mitigated the vaginal damage and reduced the secretion of proinflammatory factors (TNF-α, IL-1α and IL-β). Therefore, it is a promising therapy for the clinical treatment of VVC in the near future.

Background: Bacterial vaginosis (BV) is the most prevalent cause of abnormal vaginal discharge among pre-menopausal women and associated with adversities of sexual and reproductive health. The present study aimed to identify potential epidemiological and behavioural risk factors and clinical predictors of BV among women in Delhi, India. Methods: A cross-sectional study was conducted to assess 283 non-pregnant women aged 18-45 years for BV using Nugent's scoring criteria. Information on demographics, sexual behaviours, hygiene practices and clinical symptoms was obtained and evaluated for their association with Nugent-BV status. Results: A positive diagnosis for Nugent-BV was made in 69 (24.4%) participants, 55 (19.4%) were intermediate and 159 (65.2%) were negative for Nugent-BV. Infertility (p = .02) and recent unprotected sexual exposure (p = .02) were strongly associated with Nugent-BV. On the other hand, women who reported regular use of condoms during intercourse were more likely to test negative (p = .03). None of the patient complaints, however, had any significant correlation with Nugent-BV diagnosis. Conclusion: Women in their reproductive years share the highest burden of adversities associated with bacterial vaginosis. History of infertility, recent unprotected sexual exposure and frequent use of condoms were correlates having significant associations with Nugent-BV.

Antifungal drug susceptibility tests (AST) for Candida albicans are increasingly demanded for women with refractory or recurrent Candida vaginitis due to fluconazole resistance. Given reduced activity of azole drugs at pH levels found in women with Candida vaginitis, it is proposed that AST be performed at pH 4.5, since testing at only the recommended pH 7.0 is likely to miss a significant number of clinically relevant azole-resistant C. albicans vaginal isolates.

Bacterial vaginosis (BV) is associated with serious gynaecologic and obstetric complications. The hallmark of BV is the presence of a polymicrobial biofilm on the vaginal epithelium, but BV aetiology is still a matter of debate. We have previously developed an in vitro biofilm model that included three BV-associated species, but, up to now, no studies are available whereby more bacterial species are grown together to better mimic the in vivo situation. Herein, we characterized the first polymicrobial BV biofilm consisting of six cultivable BV-associated species by using both in vitro and ex vivo vaginal tissue models. Both models revealed that the six species were able to incorporate the polymicrobial biofilm, at different bacterial concentrations. As it has been thought that this polymicrobial biofilm may increase the survival of BV-associated species when exposed to antibiotics, we also assessed if the Thymbra capitata essential oil (EO), which has recently been shown to be highly bactericidal against several Gardnerella species, could maintain its anti-biofilm activity against this polymicrobial biofilm. Under our experimental conditions, T. capitata EO exhibited a high antibacterial effect against polymicrobial biofilms, in both tested models, with a significant reduction in the biofilm biomass and the number of culturable cells. Overall, this study shows that six BV-associated species can grow together and form a biofilm both in vitro and when using an ex vivo model. Moreover, the data obtained herein should be considered in further applications of T. capitata EO as an antimicrobial agent fighting BV.

Introduction. Two high-oncogenic-risk human papilomavirus (hrHPV) genotypes - HPV16 and HPV18 - cause most of the cases of cervical cancer worldwide. Bacterial vaginosis is associated with increased hrHPV persistence, although the mechanism underlying this association remains unclear. Gardnerella spp. are detected in nearly all cases of bacterial vaginosis and are the major source of cervicovaginal sialidases. The NanH1 gene is present in virtually all Gardnerella sialidase-producing strains and has been proposed as a potential marker for persistent hrHPV infection.Hypothesis. Gardnerella spp. load and the NanH1 gene are associated with hrHPV persistence.Aim. To compare the cervicovaginal load of Gardnerella spp. and the frequency of the NanH1 gene between women with persistent HPV16 and/or HPV18 infection and those who cleared the infection after 11 months.Methodology. Among a population of 1638 HPV screened, we detected 104 with positive HPV16 and/or HPV18 results. Samples were obtained at two time points (baseline and at a median of 11 months at follow-up) and tested using the Linear Array HPV Genotyping kit (Roche Molecular Systems, Pleasanton, CA, USA). Based on their HPV16/HPV18 status at enrolment and follow-up, participants were assigned to 'persistence' or 'clearance' groups. We used cervicovaginal fluid samples obtained upon enrolment to determine the load of the 23 s rRNA gene of Gardnerella spp. and the presence of the NanH1 gene using real-time polymerase chain reaction (PCR). We compared Gardnerella spp. loads and NanH1 frequency between the groups by, respectively, Mann-Whitney and chi-squared tests, with a P-value <0.05 considered to be significant.Results. Of the 104 participants who were positive for HPV16/HPV18, 73 (70.2 %) persisted with at least 1 of the baseline genotypes at follow-up, while 31 (29.8 %) cleared the infection in this time frame. Participants in the persistence group had significantly higher loads of Gardnerella spp. [5.8E+02 (0-3.0E+05) copies µl-1] than those in the clearance group [9.9E+01 (0-7.7E+04) copies µl-1] (P=0.03). The baseline frequency of NanH1 was higher in the persistence' (n=46, 63.0 %) than in the clearance (n=14, 45.2 %) group, although this was not statistically significant (P=0.09).Conclusion. These findings reinforce the negative effect of vaginal microbiota for the clearance of hrHPV and indicate a possible association between sialidase-producing species with hrHPV persistence.

Gardnerella vaginalis is the core pathogen of bacterial vaginosis (BV), the most common vaginal infection in women. G. vaginalis exerts pathogenicity through various factors, such as biofilm formation and the local host immune response stimulation. Therefore, this study aimed to evaluate the inhibitory effect of Lactobacillus gasseri CCFM1201 on G. vaginalis using experimental BV models. We evaluated L. gasseri in vitro to inhibit pathogen biofilm formation and adhesion capacity in HeLa cells using crystal violet staining. Further in vivo studies were conducted to assess the inhibitory effects of L. gasseri CCFM1201 on BV induced by G. vaginalis. L. gasseri exhibited strain-specific adhesion and inhibition of pathogen biofilm formation in vitro. L. gasseri CCFM1201 significantly reduced G. vaginalis in mice (p < 0.05), inhibited sialidase activity, modulated tumor necrosis factor-α and interleukin-1β expression, and reduced myeloperoxidase activity (p < 0.05). Histopathological examination indicated that L. gasseri CCFM1201 improved inflammatory cell infiltration of vaginal tissue and restored its structure. Vaginal epithelial cell exfoliation, the main clinical feature of BV, was significantly improved by L. gasseri CCFM1201 intervention (p < 0.05). Thus, L. gasseri CCFM1201 is a potential candidate for treating G. vaginalis-induced vaginal diseases.