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 vaginal microbiota and innate immunity after local excisional treatment for cervical intraepithelial neoplasia

Background Vaginal microbiota (VMB) composition is altered in women with cervical intra-epithelial neoplasia (CIN) compared to healthy controls and is associated with disease progression. However, the impact of CIN excision on the VMB and innate immunity is not known. This observational study aims to explore the impact of CIN excision on the VMB, antimicrobial peptides (AMP) and proinflammatory cytokines. Methods We sampled 103 non-pregnant, premenopausal women at the time of excisional treatment for CIN and at their 6-month follow-up visit. A further 39 untreated controls with normal cytology were also sampled. We used metataxonomics to group vaginal swab samples into community state types (CSTs) and ELISA to quantify cytokine and AMP levels in matched vaginal secretions. Analyses were performed to compare the bacterial composition and immune analyte levels before and after CIN excision and in healthy controls. Results Women with CIN had significantly higher rates of Lactobacillus species depletion pre-treatment compared to healthy controls (CST IV 21/103, 20% vs 1/39, 3%, p = 0.0081). Excision did not change the VMB composition, with CST IV remaining significantly more prevalent after excision compared to untreated, healthy controls (CST IV 19/103, 20% vs 1/39, 3%, p = 0.0142). Prevotella bivia and Sneathia amnii were significantly higher in samples before treatment compared to untreated controls, and Prevotella bivia remained significantly higher amongst the treated, with less Lactobacillus crispatus compared to untreated controls. IL-1β and IL-8 remained significantly elevated pre- (p < 0.0001 and p = 0.0014, respectively) and post-treatment (p < 0.0001 and p = 0.0035, respectively) compared to untreated controls. Levels of human beta-defensin-1 and secretory leukocyte protease inhibitor were both significantly reduced following CIN excision (p < 0.0001); however, their levels remained lower than controls post-treatment. Conclusions Women with CIN have an increased prevalence of Lactobacillus sp. depletion, high-diversity VMB composition, and higher levels of proinflammatory cytokines and AMPs compared to normal controls. Surgical excision of the disease reduces levels of vaginal AMPs but does not alter VMB composition or cytokine levels. These findings suggest that women with CIN have an inherent predisposition to a high-diversity proinflammatory environment that is not corrected by disease excision. The failure to re-establish a Lactobacillus-enriched CST may explain why women remain at high risk of pre-invasive and invasive disease recurrence.

Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

Microbiome Profiling Using Shotgun Metagenomic Sequencing Identified Unique Microorganisms in COVID-19 Patients With Altered Gut Microbiota

COVID-19 is mainly associated with respiratory distress syndrome, but a subset of patients often present gastrointestinal (GI) symptoms. Imbalances of gut microbiota have been previously linked to respiratory virus infection. Understanding how the gut–lung axis affects the progression of COVID-19 can provide a novel framework for therapies and management. In this study, we examined the gut microbiota of patients with COVID-19 (n = 47) and compared it to healthy controls (n = 19). Using shotgun metagenomic sequencing, we have identified four microorganisms unique in COVID-19 patients, namely Streptococcus thermophilus, Bacteroides oleiciplenus, Fusobacterium ulcerans, and Prevotella bivia. The abundances of Bacteroides stercoris, B. vulgatus, B. massiliensis, Bifidobacterium longum, Streptococcus thermophilus, Lachnospiraceae bacterium 5163FAA, Prevotella bivia, Erysipelotrichaceae bacterium 6145, and Erysipelotrichaceae bacterium 2244A were enriched in COVID-19 patients, whereas the abundances of Clostridium nexile, Streptococcus salivarius, Coprococcus catus, Eubacterium hallii, Enterobacter aerogenes, and Adlercreutzia equolifaciens were decreased (p &lt; 0.05). The relative abundance of butyrate-producing Roseburia inulinivorans is evidently depleted in COVID-19 patients, while the relative abundances of Paraprevotella sp. and the probiotic Streptococcus thermophilus were increased. We further identified 30 KEGG orthology (KO) modules overrepresented, with 7 increasing and 23 decreasing modules. Notably, 15 optimal microbial markers were identified using the random forest model to have strong diagnostic potential in distinguishing COVID-19. Based on Spearman’s correlation, eight species were associated with eight clinical indices. Moreover, the increased abundance of Bacteroidetes and decreased abundance of Firmicutes were also found across clinical types of COVID-19. Our findings suggest that the alterations of gut microbiota in patients with COVID-19 may influence disease severity. Our COVID-19 classifier, which was cross-regionally verified, provides a proof of concept that a set of microbial species markers can distinguish the presence of COVID-19.

Vaginal Microbiota of the Sexually Transmitted Infections Caused by Chlamydia trachomatis and Trichomonas vaginalis in Women with Vaginitis in Taiwan

The three most common sexually transmitted infections (STIs) are Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC) and Trichomonas vaginalis (TV). The prevalence of these STIs in Taiwan remains largely unknown and the risk of STI acquisition affected by the vaginal microbiota is also elusive. In this study, a total of 327 vaginal swabs collected from women with vaginitis were analyzed to determine the presence of STIs and the associated microorganisms by using the BD Max CT/GC/TV molecular assay, microbial cultures, and 16S rRNA sequencing. The prevalence of CT, TV, and GC was 10.8%, 2.2% and 0.6%, respectively. A culture-dependent method identified that Escherichia coli and Streptococcus agalactiae (GBS) were more likely to be associated with CT and TV infections. In CT-positive patients, the vaginal microbiota was dominated by L. iners, and the relative abundance of Gardnerella vaginalis (12.46%) was also higher than that in TV-positive patients and the non-STIs group. However, Lactobacillus spp. was significantly lower in TV-positive patients, while GBS (10.11%), Prevotella bivia (6.19%), Sneathia sanguinegens (12.75%), and Gemella asaccharolytica (5.31%) were significantly enriched. Using an in vitro co-culture assay, we demonstrated that the growth of L. iners was suppressed in the initial interaction with TV, but it may adapt and survive after longer exposure to TV. Additionally, it is noteworthy that TV was able to promote GBS growth. Our study highlights the vaginal microbiota composition associated with the common STIs and the crosstalk between TV and the associated bacteria, paving the way for future development of health interventions targeting the specific vaginal bacterial taxa to reduce the risk of common STIs.

Vaginal biocenosis in patients with gynecological cancers.

e17574 Background: The purpose of the study was to assess vaginal biocenosis in patients with gynecological cancers (GCs) and in non-cancer women. Methods: The study included 50 patients aged 52.3+2 years with histologically verified GCs (cervical cancer – 23, endometrial cancer – 27) – group 1, and 22 non-cancer women aged 42.4+2.3 – group 2. Vaginal swabs were taken prior to antitumor treatment. Real-time PCR was used to determine the total bacteria mass (TBM) and DNAs of 16 groups of microorganisms: Lactobacillus spp., Enterobacteriaceae, Streptococcus spp., Staphylococcus spp., Gardnerella vaginalis+Prevotella bivia+Porphyromonas spp., Eubacterium spp., Sneathia spp.+Leptotrichia spp.+Fusobacterium spp., Megasphaera spp.+Veillonella spp.+Dialister spp., Lachnobacterium spp.+Clostridium spp., Mobiluncus spp.+Corynebacterium spp., Peptostreptococcus spp., Atopobium vaginae, Candida spp., Mycoplasma hominis, Ureaplasma (urealyticum+ parvum), Mycoplasma genitalium. Results: Vaginal biocenosis was assessed as normocenosis in 12 (24%) women in group 1 and 12 (54.5%, p = 0.012) women in group 2; conditional normocenosis - 2 (4%) and 3 (13.6%, p > 0.05); dysbiosis - 36 (72%) and 7 (31.8%, p = 0.0017), respectively. Anaerobic bacteria prevailed in women with dysbiosis in both groups (46% and 22.7%, p = 0.005); aerobic dysbiosis was registered in 16% and 4.5% (p > 0.05), mixed dysbiosis – in 10% and 4.5% (p > 0.05) of women of groups 1 and 2, respectively. Normal flora ( Lactobacillus spp.) was observed in 23 (46.0%) in group 1 and 17 (77.3%, p = 0.02) in group 2. Streptococcus spp. >10% of TBM were found in 9 (21.9%) women in group 1 and not found in group 2 (p = 0.049). Gardnerella vaginalis+Prevotella bivia+Porphyromonas spp. >10% of TBM were found in 27 (54.0%) in group 1 and 6 (27.3%, p = 0.031) in group 2; Staphylococcus spp. - 13 (26.0%) in group 1 and 12 (54.5%, p = 0.031) in group 2. Conclusions: Development of gynecological tumors was accompanied by a sharp decrease in Lactobacillus spp., a significant increase in Gardnerella vaginalis+ Prevotella bivia+ Porphyromonas spp. and Streptococcus spp., vaginal dysbiosis with a predominance of anaerobic and mixed microflora.

Atopobium vaginae and Prevotella bivia Are Able to Incorporate and Influence Gene Expression in a Pre-Formed Gardnerella vaginalis Biofilm

Bacterial vaginosis (BV) is associated with a highly structured polymicrobial biofilm on the vaginal epithelium where Gardnerella species presumably play a pivotal role. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia are vaginal pathogens detected during the early stages of incident BV. Herein, we aimed to analyze the impact of A. vaginae and P. bivia on a pre-established G. vaginalis biofilm using a novel in vitro triple-species biofilm model. Total biofilm biomass was determined by the crystal violet method. We also discriminated the bacterial populations in the biofilm and in its planktonic fraction by using PNA FISH. We further analyzed the influence of A. vaginae and P. bivia on the expression of key virulence genes of G. vaginalis by quantitative PCR. In our tested conditions, A. vaginae and P. bivia were able to incorporate into pre-established G. vaginalis biofilms but did not induce an increase in total biofilm biomass, when compared with 48-h G. vaginalis biofilms. However, they were able to significantly influence the expression of HMPREF0424_0821, a gene suggested to be associated with biofilm maintenance in G. vaginalis. This study suggests that microbial relationships between co-infecting bacteria can deeply affect the G. vaginalis biofilm, a crucial marker of BV.

Changes in the Vaginal Microbiome during the Pregnancy to Postpartum Transition

Substantial changes in the composition of the vaginal microbiome occur following the end of pregnancy. To identify potential drivers of microbiome changes in individual women during the pregnancy to postpartum transition, we evaluated vaginal samples from 48 pregnant women during their first and third trimesters and postpartum. We determined the species composition of vaginal communities and the vaginal fluid levels of compounds involved in mediating changes in host physiology and the immune system at each time point. We used linear mixed-effects models to characterize associations. Consistent with previous reports, but with a larger sample size, a US population, and variations in the dominant bacteria, the vaginal microbiome was found to be more diverse during the postpartum period. There was a lower abundance of Lactobacillus and significantly higher proportions of Streptococcus anginosus and Prevotella bivia. Moreover, we uniquely demonstrated that postpartum vaginal secretions were also altered postpartum. There were elevated levels of hyaluronan and Hsp70 and decreased levels of the D- and L-lactic acid isomers. We posit that these variations are consequences of alterations in the vagina after delivery that profoundly alter the host environment and, thus, lead to changes in the capability of different bacterial species to survive and proliferate.