A cultural microbiological study of the vaginal microbiota of patients of reproductive age was carried out to isolate the species Lactobacillus iners with subsequent study of phenotypic features. The presence of two phenotypically different species variants was found in patients with bacterial vaginosis.

Bacterial vaginosis (BV) is a most common microbiological syndrome. Multiplex next-generation sequencing (NGS) or molecular tests allow a complete and accurate vaginal microbiota profiling in order to determine the primary causative agent. Due to the high costs and limited availability of NGS, the multiplex real-time PCR draws more attention. The present study aimed to evaluate the microbial composition and dominant lactobacilli species in non-pregnant women with bacterial vaginosis using a multiplex RT-PCR test and determine its diagnostic significance. In total, 331 women complaining of vaginal discharge were included. BV was confirmed upon clinical examination and Nugent criteria. A real-time PCR test was carried out with a new Femoflor test, which identifies opportunistic bacteria, STD pathogens, and some viruses. According to the results, the rate of lactobacilli is significantly reduced in BV-affected patients when compared to healthy women. Moreover, the rate of L. crispatus significantly decreases, while the rate of L. iners remains high. Among obligate anaerobic bacteria, Gardnerella vaginalis was the most prevalent in women with BV. The Femoflor test demonstrated high sensitivity and specificity for diagnosing BV. Moreover, the test allows the identification of infection in women with intermediate vaginal microbiota, as well as STD pathogens, and viruses. Thus, the application of real-time PCR tests can be effectively used in vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the Amsel criteria and Nugent scoring method in diagnosing BV.

Emerging evidence suggests that the reproductive tract microbiota is a key modulator of local inflammatory and immune pathways throughout pregnancy and may subsequently impact pregnancy outcomes. In this study, our objective was to analyze the cervical and vaginal microbiomes during early pregnancy among three groups: women with healthy ongoing pregnancies, women undergoing dydrogesterone treatment, and those who experienced miscarriages. The experiment involved 51 women at 8-11 weeks of gestation. The microbiome was examined using 16S rRNA sequencing on the Ion Torrent PGM platform. Across all groups, Lactobacillus iners was predominant, suggesting that the vaginal community type CST III is common among the majority of participants. Notably, our data highlighted the significant roles of Gardnerella vaginalis and Mycoplasma girerdii in the pathogenesis of early miscarriage. Conversely, L. iners and Bifidobacterium longum have a protective effect in early pregnancy. Moreover, dydrogesterone intake appeared to influence notable differences between the cervical and vaginal microbiomes. Overall, our study enhanced our understanding of the cervical and vaginal microbiome composition in the eastern European population during early pregnancy.

UNASSIGNED: Vaginal lactobacilli are recognized as important drivers of genital health including protection against bacterial vaginosis and sexually transmitted infections. Lactobacillus iners is distinct from L. crispatus, L. gasseri, and L. jensenii by its high global prevalence in vaginal microbiomes, relatively small genome, production of only L-lactic acid, and inconsistent associations with genital health outcomes. In this review, we summarize our current understanding of the role of L. iners in the vaginal microbiome, highlight the importance of strain-level consideration for this species, and explain that while marker gene-based characterization of the composition of the vaginal microbiota does not capture strain-level resolution, whole metagenome sequencing can aid in expanding our understanding of this species in genital health.
UNASSIGNED: L. iners exists in the vaginal microbiome as a unique combination of strains. The functional repertoires of these strain combinations are likely wide and contribute to the survival of this species in a variety of vaginal microenvironments. In published studies to date, strain-specific effects are aggregated and may yield imprecise estimates of risk associated with this species.
UNASSIGNED: The worldwide high prevalence of Lactobacillus iners warrants more research into its functional roles in the vaginal microbiome and how it may directly impact susceptibility to infections. By incorporating strain-level resolution into future research endeavors, we may begin to appreciate L. iners more thoroughly and identify novel therapeutic targets for a variety of genital health challenges.

Several studies have compared metagenome inference performance in different human body sites; however, none specifically reported on the vaginal microbiome. Findings from other body sites cannot easily be generalized to the vaginal microbiome due to unique features of vaginal microbial ecology, and investigators seeking to use metagenome inference in vaginal microbiome research are \"flying blind\" with respect to potential bias these methods may introduce into analyses. We compared the performance of PICRUSt2 and Tax4Fun2 using paired 16S rRNA gene amplicon sequencing and whole-metagenome sequencing data from vaginal samples from 72 pregnant individuals enrolled in the Pregnancy, Infection, and Nutrition (PIN) cohort. Participants were selected from those with known birth outcomes and adequate 16S rRNA gene amplicon sequencing data in a case-control design. Cases experienced early preterm birth (<32 weeks of gestation), and controls experienced term birth (37 to 41 weeks of gestation). PICRUSt2 and Tax4Fun2 performed modestly overall (median Spearman correlation coefficients between observed and predicted KEGG ortholog [KO] relative abundances of 0.20 and 0.22, respectively). Both methods performed best among Lactobacillus crispatus-dominated vaginal microbiotas (median Spearman correlation coefficients of 0.24 and 0.25, respectively) and worst among Lactobacillus iners-dominated microbiotas (median Spearman correlation coefficients of 0.06 and 0.11, respectively). The same pattern was observed when evaluating correlations between univariable hypothesis test P values generated with observed and predicted metagenome data. Differential metagenome inference performance across vaginal microbiota community types can be considered differential measurement error, which often causes differential misclassification. As such, metagenome inference will introduce hard-to-predict bias (toward or away from the null) in vaginal microbiome research. IMPORTANCE Compared to taxonomic composition, the functional potential within a bacterial community is more relevant to establishing mechanistic understandings and causal relationships between the microbiome and health outcomes. Metagenome inference attempts to bridge the gap between 16S rRNA gene amplicon sequencing and whole-metagenome sequencing by predicting a microbiome\'s gene content based on its taxonomic composition and annotated genome sequences of its members. Metagenome inference methods have been evaluated primarily among gut samples, where they appear to perform fairly well. Here, we show that metagenome inference performance is markedly worse for the vaginal microbiome and that performance varies across common vaginal microbiome community types. Because these community types are associated with sexual and reproductive outcomes, differential metagenome inference performance will bias vaginal microbiome studies, obscuring relationships of interest. Results from such studies should be interpreted with substantial caution and the understanding that they may over- or underestimate associations with metagenome content.

Vaginal infections continue to be a serious public health issue, and developing new approaches to address antibiotic-resistant pathogens is an urgent task. The dominant vaginal Lactobacillus species and their active metabolites (e.g., bacteriocins) have the potential to defeat pathogens and help individuals recover from disorders. Here, we describe for the first time a novel lanthipeptide, inecin L, a bacteriocin from Lactobacillus iners with posttranslational modifications. The biosynthetic genes of inecin L were actively transcribed in the vaginal environment. Inecin L was active against the prevailing vaginal pathogens, such as Gardnerella vaginalis and Streptococcus agalactiae, at nanomolar concentrations. We demonstrated that the antibacterial activity of inecin L was closely related to the N terminus and the positively charged His13 residue. In addition, inecin L was a bactericidal lanthipeptide that showed little effect on the cytoplasmic membrane but inhibited the cell wall biosynthesis. Thus, the present work characterizes a new antimicrobial lanthipeptide from a predominant species of the human vaginal microbiota. IMPORTANCE The human vaginal microbiota plays essential roles in preventing pathogenic bacteria, fungi, and viruses from invading. The dominant vaginal Lactobacillus species show great potential to be developed as probiotics. However, the molecular mechanisms (such as bioactive molecules and their modes of action) involved in the probiotic properties remain to be determined. Our work describes the first lanthipeptide molecule from the dominant Lactobacillus iners. Additionally, inecin L is the only lanthipeptide found among the vaginal lactobacilli thus far. Inecin L shows strong antimicrobial activity toward the prevalent vaginal pathogens and antibiotic-resistant strains, suggesting that inecin L is a potent antibacterial molecule for drug development. In addition, our results show that inecin L exhibits specific antibacterial activity related to the residues in the N-terminal region and ring A, which will contribute to structure-activity relationship studies in lacticin 481-like lanthipeptides.

The composition of the vaginal microbiome may lead to adverse pregnancy outcomes. Normal pregnancy is associated with changes in the vaginal bacterial community composition, which tend to be more enriched with one or two Lactobacillus species promoting a healthy vagina and favorable birth outcomes. The aim of the current study was to determine compositional changes in the healthy vaginal microbiome composition during the three trimesters of pregnancy in Ismailia, Egypt using Illumina MiSeq sequencing of the V3-V4 region of the 16S rRNA. The phylum Firmicutes and the genus Lactobacillus dominated across the three trimesters of pregnancy. L. iners was the most abundant species. However, L. coleohominis and L. reuteri represented the least dominant vaginal lactobacilli. Core microbiome analyses showed the Lactobacillus genus and L. iners species to have the highest prevalence in all the samples of our study groups. The phylum Firmicutes was found to be negatively correlated with almost all other vaginal phyla during pregnancy. Likewise, a negative correlation between Lactobacillus and almost all other genera was detected, including significant negative correlations with Dialister and Prevotella. Furthermore, negative correlations of L. iners were detected with almost all other species, including a significant negative correlation with L. helveticus, G. vaginalis, S. anginosus, and S. agalactiae.

Few studies have investigated the vaginal microbiota (VM) in women living with HIV (WLWH) in the context of high-risk human papillomavirus (HR-HPV) infection, even though WLWH are at an increased risk of HPV-related malignancies, including cervical cancer. To explore the impact of HIV and HPV infection on the VM in WLWH, we determined the prevalence of HR-HPV infection and cervical cytologic abnormalities in a cohort of 44 WLWH and 39 seronegative-women (SNW), characterized the vaginal microbiota by 16S sequencing, assessed genital inflammation and systemic immune activation by multiplex bead assay and flow cytometry, respectively. Finally, we explored relationships between bacterial richness and diversity, the top 20 bacterial genera, genital inflammation and systemic immune activation.
We found that HR-HPV prevalence was similar between WLWH and SNW. High-grade squamous intraepithelial lesions (HSIL) were only detected in WLWH negative for HR-HPV infection. In regression analyses, no risk factors were identified. Women co-infected with HIV and HR-HPV had the highest level of systemic immune activation, and these levels were significantly different compared with SNW without HR-HPV infection. Lactobacillus iners was the dominant Lactobacillus species in WLWH and SNW alike.
We found no evidence of differences in vaginal microbial richness and diversity, microbial community structure, and genital inflammation by HIV, HPV, or HIV and HPV status.

In reproductive-age women, the vaginal microbiome is typically dominated by one or a few Lactobacillus species, including Lactobacillus crispatus, Lactobacillus iners, Lactobacillus paragasseri, Lactobacillus mulieris, and Lactobaccillus crispatus, has been associated with optimal cervicovaginal health; however, much is still unknown about how other lactobacilli metabolically contribute to cervicovaginal health. We hypothesized that metabolites of each Lactobacillus species differ and uniquely contribute to health and homeostasis. To address this hypothesis, we utilized a human three-dimensional (3D) cervical epithelial cell model in conjunction with genomics analyses and untargeted metabolomics to determine the metabolic contributions of less-studied vaginal lactobacilli-L. iners, L. paragasseri, and L. mulieris. Our study validated that vaginal lactobacilli exhibit a close phylogenetic relationship. Genomic findings from publicly available strains and those used in our study indicated that L. iners is metabolically distinct from other species of lactobacilli, likely due to a reduced genome size. Lactobacilli and mock controls were distinguishable based on global metabolic profiles. We identified 95 significantly altered metabolites (P < 0.05) between individual lactobacilli and mock controls. Metabolites related to amino acid metabolism were shared among the lactobacilli. N-Acetylated amino acids with potential antimicrobial properties were significantly elevated in a species-specific manner. L. paragasseri and L. iners shared aromatic, but not carbohydrate-derived, lactic acid metabolites with potential antimicrobial properties that may contribute to homeostasis of the cervicovaginal environment. Additionally, L. iners uniquely altered lipid metabolism, which may be a sign of adaptation to the cervicovaginal niche. Overall, these findings further elucidate the metabolic contributions of three key vaginal Lactobacillus species in gynecological health. IMPORTANCE Lactobacillus species contribute to cervicovaginal health by their production of lactic acid and other antimicrobial compounds. Yet, much is still unknown regarding the metabolic potential of lesser-studied but common vaginal lactobacilli. Here, we used untargeted metabolomics coupled with our 3D cervical epithelial cell model to identify metabolic differences among vaginal Lactobacillus species (Lactobacillus iners, Lactobacillus paragasseri, and Lactobacillus mulieris) and how those differences related to maintaining homeostasis of the cervical epithelium. Human 3D cell models are essential tools for studying host-bacteria interactions and reducing confounding factors inherent in clinical studies. Therefore, these unique models allowed us to decipher the putative lactobacilli mechanisms that contribute to their roles in health or disease. Metabolic analyses revealed distinct profiles of each Lactobacillus species but also shared metabolic contributions associated with antimicrobial activity: amino acid metabolism, N-acetylated amino acids, and aromatic lactic acids. These patterns provided validation of metabolites associated with health in clinical studies and provided novel targets, including immunomodulatory and antimicrobial metabolites, for postbiotic therapies.