Role of lactobacilli and lactoferrin in the mucosal cervicovaginal defense

Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by cholangiocyte death and ductopenia

Characterization of the vaginal microbiome in pregnancy

The vaginal microbiome plays an important role in women's reproductive health. Imbalances in this microbiota are associated with bacterial vaginosis, increased susceptibility to sexually transmitted infections, and negative reproductive outcomes. The causes of such variations, however, are poorly understood. A healthy vaginal microbiota is defined as Lactobacillus-dominated and an overgrowth of other species is often associated with unhealthy conditions. An appreciation of "atypical" microbiomes in healthy women, such as Bifidobacterium-dominated, has been gradually increasing. Although bifidobacteria play an important role in gut health, vaginal bifidobacteria have not yet been fully characterized. In this study, a baseline description of the "healthy" vaginal microbiome in pregnancy has been established based on cpn60 gene amplicon sequencing. The vaginal microbiota of pregnant women relative to non-pregnant women had lower richness and diversity, higher Lactobacillus abundance and lower Mollicutes/Ureaplasma prevalence. This gives a better understanding of the vaginal microbiome in healthy pregnancies and provides a control group for a subsequent comparison to women who experienced preterm birth. An association between Mollicutes and preterm was confirmed, and further suggested that a more rich and diverse microbiome is associated with prematurity. To better understand the relationship between reproductive outcomes and microbiota, an improved definition of the healthy microbiome is also needed, which should include evaluation of "atypical" microbiomes, such as Bifidobacterium-dominated. Phenotypic characterization of vaginal bifidobacteria indicated that they have health promoting characteristics similar to beneficial vaginal lactobacilli. Considering the importance of bifidobacteria as one of the primary colonizers of the neonatal gut, the genomes of vaginal and gut isolates of Bifidobacterium breve and Bifidobacterium longum were compared. Results indicated that vaginal and gut microbiomes are colonized by a shared community of Bifidobacterium, which may be transferred from mother to infant. Taken together, the results presented in this thesis provide a better understanding of the vaginal microbiome of pregnant women with low and high risk for preterm birth. It also improves the understanding of a healthy microbiome by phenotypically characterizing vaginal bifidobacteria, and contributes to elucidate aspects of bifidobacteria ecology by comparing the genomes of vaginal and gut bifidobacteria

Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria

Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV.Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel's clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel's criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV.The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in Black women. Tools developed in this project can be used to study microbial ecology in diverse settings at high resolution

Probiotic Therapy for the Prevention of Recurrent Spontaneous Preterm Birth

Preterm birth is the national leading cause of neonatal morbidity and mortality. Nearly 500,000 infants are born prematurely each year in the United States and the annual cost of prematurity averages over $26 billion dollars. Despite multiple interventions to reduce infection related prematurity many spontaneous preterm births remain attributed infection and inflammation. We hypothesize that prevention of infection and inflammation related spontaneous preterm birth is possible through enrichment, stabilization, and normalization of the vaginal microbiome. To test this hypothesis we will perform a randomized, double blind, placebo-controlled trial of intravaginal Lactobacillus probiotic in women at high-risk for spontaneous preterm birth. Primary outcome will be reduction frequency of spontaneous preterm birth prior to 37 weeks completed gestation. Results of this study will heighten understanding of the role of the normal vaginal flora in pregnancy and potentially uncover a new intervention capable of reducing preterm birth and the complications of prematurity

Characterizing the human vaginal microbiome using high-throughput sequencing

The human vaginal microbiome undoubtedly has a significant role in reproductive health and for protection from infectious organisms. Recent efforts to characterize the bacterial species of the vagina using molecular techniques have uncovered an unexpected diversity. Using high-throughput sequencing I sought to describe the structure and function of the vaginal microbiome under different physiological states including healthy, bacterial vaginosis (BV), post-menopausal vaginal atrophy, and acute vulvovaginal candidiasis (VVC). Partial 16S rRNA gene sequencing revealed that healthy, asymptomatic women most often have vaginal biotas dominated by Lactobacillus iners or L. crispatus. In contrast, BV is a heterogeneous, highly diversified condition with reduced Lactobacillus abundance. Similar to BV, post-menopausal women experiencing vaginal dryness were depleted in lactobacilli and had a more diverse vaginal profile. In the case of VVC, the biotas were not significantly altered compared to healthy women despite the fungal overgrowth. One organism, Lactobacillus iners was ubiquitously present in all conditions, and became predominant following antibiotic and probiotic treatment of BV. To uncover the potential role of this bacterium, I used whole genome sequencing of vaginal isolate AB-1. The genome is predicted to be the smallest of any Lactobacillus at 1.3 Mbp, but having a higher proportion of horizontally acquired genes. These results, along with predicted adhesins and a cholesterol-dependent cytolysin, indicate L. iners is highly adapted for the vagina and could have an uncharacterized role in the etiology of BV. As BV is the most common vaginal ailment with severe implications on acquisition and transmission of sexually transmitted infections, and complications during pregnancy, I sought iito examine the functional contribution of the organisms during BV using meta-RNA sequencing. L. iners drastically modulates gene expression in response to BV, and notably increases expression of a cholesterol-dependent cytolysin, mucin and glycerol transport and metabolic enzymes, and genes belonging to a CRISPR system - suggestive of bacteriophage influence in the community. Although diverse in taxonomic membership, there is evidence of functional conservation in BV including preference for glycogen and glycerol as carbon sources, and predicted end products of metabolism including an abundance of succinate and short-chain fatty acids. These studies add significantly to our understanding of the role lactobacilli can play in vaginal and reproductive health

Profiles and technological requirements of urogenital probiotics

Probiotics, defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, are considered a valid and novel alternative for the prevention and treatment of female urogenital tract infections. Lactobacilli, the predominant microorganisms of the healthy human vaginal microbiome, can be included as active pharmaceutical ingredients in probiotics products. Several requirements must be considered or criteria fulfilled during the development of a probiotic product or formula for the female urogenital tract. This review deals with the main selection criteria for urogenital probiotic microorganisms: host specificity, potential beneficial properties, functional specifications, technological characteristics and clinical trials used to test their effect on certain physiological and pathological conditions. Further studies are required to complement the current knowledge and support the clinical applications of probiotics in the urogenital tract. This therapy will allow the restoration of the ecological equilibrium of the urogenital tract microbiome as well as the recovery of the sexual and reproductive health of women.Fil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Juárez Tomás, María Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentin

Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin

© 2018 The Author(s). Background: Preterm prelabour rupture of the fetal membranes (PPROM) precedes 30% of preterm births and is a risk factor for early onset neonatal sepsis. As PPROM is strongly associated with ascending vaginal infection, prophylactic antibiotics are widely used. The evolution of vaginal microbiota compositions associated with PPROM and the impact of antibiotics on bacterial compositions are unknown. Methods: We prospectively assessed vaginal microbiota prior to and following PPROM using MiSeq-based sequencing of 16S rRNA gene amplicons and examined the impact of erythromycin prophylaxis on bacterial load and community structures. Results: In contrast to pregnancies delivering at term, vaginal dysbiosis characterised by Lactobacillus spp. depletion was present prior to the rupture of fetal membranes in approximately a third of cases (0% vs. 27%, P=0.026) and persisted following membrane rupture (31%, P=0.005). Vaginal dysbiosis was exacerbated by erythromycin treatment (47%, P=0.00009) particularly in women initially colonised by Lactobacillus spp. Lactobacillus depletion and increased relative abundance of Sneathia spp. were associated with subsequent funisitis and early onset neonatal sepsis. Conclusions: Our data show that vaginal microbiota composition is a risk factor for subsequent PPROM and is associated with adverse short-term maternal and neonatal outcomes. This highlights vaginal microbiota as a potentially modifiable antenatal risk factor for PPROM and suggests that routine use of erythromycin for PPROM be re-examined

Resolution and characterization of subgroups of Gardnerella vaginalis and description of the vaginal microbiota of women with preterm premature rupture of membranes

The vaginal microbial community is critical to a woman’s health and the health of her family. Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a shift of the vaginal microbiota from a Lactobacillus dominated community to a dense biofilm containing a complex mixture of organisms. Although BV is an important risk factor for poor reproductive health outcomes, the etiology of BV is poorly understood. Gardnerella vaginalis is a hallmark species of BV. Phylogenetic analysis of cpn60 universal target sequences from metagenomic studies of the vaginal microbiome and from G. vaginalis isolates resolved four subgroups within the species. This subdivision, supported by whole genome similarity comparisons, demonstrated that these subgroups might represent different species. Among a group of African women, only G. vaginalis subgroup B was significantly more abundant in women with BV relative to women with Nugent scores not consistent with BV. To characterize the subgroups further, several phenotypic and molecular factors of G. vaginalis subgroups were assessed. Proteomic profiles of isolates within each subgroup formed unambiguous clusters. Sialidase gene sequences were detected in all subgroups, however enzymatic activity was detected only in subgroup B. Two isolates of subgroup B isolates (N153 and N101) were incapable of growth in 7% CO2. Given the well-known relationship between an anaerobic microbiota and BV, anaerobic isolates of G. vaginalis are potentially important players in the vaginal microbial community. To determine genome content differences that could account for the phenotypic difference, whole genome sequences of four G. vaginalis subgroup B isolates representing facultative and anaerobic phenotypes were determined. Comparison of genomes led to the identification of genes predicted to encode proteins involved in cell wall biogenesis and protection from oxidative damage that might account for the observed phenotypes. The cpn60 universal target based methodology that improved resolution of the vaginal microbiota including G. vaginalis was applied in a prospective study of the vaginal microbiome of women with preterm premature rupture of membranes (PPROM). The objectives were to characterize the vaginal microbiota of women following PPROM, and to determine if microbiome composition at the time of rupture predicts latency duration and perinatal outcomes. Only 13/70 samples collected from 36 women were dominated by Lactobacillus spp., the expected profile for healthy women, while Megasphaera type 1 and Prevotella spp. were detected in all samples. Microbiome profiles at the time of membrane rupture did not cluster by gestational age at PPROM, or latency duration. Microbial profiles were unstable over the latency period, with dramatic shifts in composition between weekly samples, and an overall decrease in Lactobacillus abundance. Mollicutes were detected by PCR in 81% (29/36) of women, and these women had significantly lower gestational age at delivery and correspondingly lower birth weight infants than Mollicutes negative women. Taken together, the results presented in this thesis demonstrate the value of high resolution profiling of the vaginal microbiome using cpn60 UT sequences. The resolution of subgroups within G. vaginalis has potentially significant implications for women's health diagnostics, requiring a shift away from considering G. vaginalis as a single entity. The PPROM study provides foundational information that may lead to the identification of informative sequence patterns, providing clinicians with better tools for expectant management following PPROM

Probiotic lactobacilli in formulas and hygiene products for the health of the urogenital tract

Lactobacilli are the predominant microorganisms of the healthy human vagina. A novel alternative for the prevention and treatment of female urogenital tract infections (UGTI) is the inclusion of these microorganisms as active pharmaceutical ingredients in probiotic formulas, and more recently in female hygienic products. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” A list of requirements must be considered during the development of probiotic product/formula for the female urogenital tract (UGT). This review aims to resume the requirements, probiotic characteristics, and clinical trial applied to determine the effect of probiotic and potentially probiotic strains on different woman’s physiological and pathological conditions, and in preterm birth prevention. A revision of female hygienic products available in the world market is included, together with novel studies applying nanotechnology for Lactobacillus incorporation in hygienic products. Further studies and well-designed clinical trials are urgently required to complement the current knowledge and applications of probiotics in the female UGT. The use of probiotic formulas and products will improve and restore the ecological equilibrium of the UGT microbiome to prevent and treat UGTI in women under different conditions.Fil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: de Gregorio, Priscilla Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Noa Sur. Instituto Superior de Investigaciones Biológicas. Grupo de Investigación y Desarrollo del Noroeste Argentino | Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas. Grupo de Investigación y Desarrollo del Noroeste Argentino; ArgentinaFil: Silva, Jessica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Microbiología e Inmunología; Argentin