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Accumulating evidence indicates the potential correlation between the vaginal microbioma and the acquisition and persistence of human papillomavirus (HPV) infection. This study aims to demonstrate the potential relationship through a systematic review of the current literature. A search was conducted on the following medical databases: PubMed and Scopus. Nineteen studies met the inclusion criteria and were incorporated in the present review. A total of 12.204 patients and their demographic characteristics were studied. Commercially available DNA tests and polymerase chain reaction (PCR) were used for the detection of different HPV subtypes, while the identification of the microbiomes was performed through specific diagnostic methods and PCR assay. The most frequently encountered species were classified based on their protective or detrimental impact on the progression of HPV infection. The beneficial role of some types of Lactobacillus (Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus crispatus) is generally supported. On the other hand, high microbial diversity and specific microorganisms such as Sneathia, Anaerococcus tetradius, Peptostreptococcus, Fusobacterium and Gardnerella vaginalis were found to be implicated with higher frequency and severity of disease, potentially resulting in pre-cancerous and cancerous cervical lesions.The role of vaginal microbiota appears to play an as yet not fully understood role in the susceptibility to HPV infection and its natural history.

BACKGROUND Vaginal lubricants are commonly used during gynecological examinations, during sexual activities, or to alleviate vaginal dryness. Many lubricants contain potentially bacteriostatic or bactericidal agents (parabens, chlorhexidine gluconate, nonoxynol-9). Our objective was to evaluate the impact of lubricants that vary in formulation on the growth and viability of vaginal Lactobacillus species and vaginal epithelial cell (VEC) colonization in an in vitro model. METHODS Growth curve, disk diffusion, and minimal inhibitory assays were used to determine the impact of lubricants or excipients on the growth of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus iners. L. crispatus strain was used in VEC colonization assays. Statistical differences were determined by analysis of variance. RESULTS Lubricants containing chlorhexidine gluconate or nonoxynol-9 (N-9; Conceptrol, K-Y Jelly, and Surgilube) significantly inhibited Lactobacillus species growth (P < 0.05). In contrast, other clinical lubricants (E-Z Lubricating Jelly, McKesson Lubricating) and personal lubricants (Astroglide Liquid, Good Clean Love Almost Naked, K-Y Warming Jelly) did not exhibit this effect. Chlorhexidine gluconate had a detrimental effect on Lactobacillus growth and exhibited stronger antimicrobial activity compared with methylparaben and propylparaben (P < 0.0001). There were lubricants that did not induce cytotoxicity in VEC (Good Clean Love Almost Naked, E-Z Lubricating Jelly, McKesson Lubricating Jelly), but these products did substantially decrease the attachment of L. crispatus to VEC, particularly when VEC were preexposed to lubricants before inoculation with bacteria (P < 0.0001). CONCLUSIONS This in vitro model indicates that select vaginal lubricants, particularly those with chlorhexidine gluconate, have potentially adverse effects on women's health by reducing growth and recolonization of vaginal Lactobacillus species.

Diagnosis of bacterial vaginosis (BV) in resource-poor settings relies on semiquantitative microscopy algorithm such as the Nugent score (NS). We evaluated a quantitative real-time PCR (qPCR) assay to detect and quantify individual BV-associated bacterial communities. Vaginal swabs from 247 South African women attending an STI clinic were evaluated for BV using NS. We used qPCR to analyze DNA from vaginal swabs for eight BV-associated bacteria, Gardnerella vaginalis (GV), Prevotella bivia (PB), BV-associated bacteria 2 (BVAB2), Megasphaera-1 (M-1), Atopobium vaginae (AV), Lactobacillus crispatus (LC), Lactobacillus jensenii (LJ), and Lactobacillus iners (LI). Sensitivities and specificities were generated for each qPCR assay. Using a ROC analysis, cutoffs were calculated for each bacterial species. A logistic regression model was used to determine the strongest predictors of BV status. Nugent scores indicated 35.6% of patients harbor BV-associated flora (NS 7-10). AV, GV, GAMB (GV + AV + M-1 + BVAB2), and LC + LJ showed the highest AUC, sensitivities, and specificities (listed respectively): AV (0.96; 96%; 93%), GV (0.88; 78%; 79%), GAMB (0.9; 87%; 82%), and LC + LJ (0.84; 82%; 72%) (all p

Introduction: The existence of a placental microbiome would require a non-antagonistic relationship between potentially colonizing bacteria and trophoblasts. Objective: The immunologic response of trophoblasts to specific potentially invading bacteria needs further analysis. Methodology: Immortalized first trimester human trophoblasts Swan 71 (Sw.71) were coincubated with Escherichia coli, Lactobacillus jensenii, Lactobacillus crispatus, and incubated alone (i.e., control group; 4 conditions with n = 6 for each condition). Chemokines and cytokines were measured. ANOVA with post hoc pairwise analysis was used to compare cytokines/chemokines concentrations in the 4 culture media. Results: Sw.71 co-incubated with E. coli, L. jensenii or L. crispatus resulted in differential secretion of 11 of the 26 assayed cytokines/chemokines. Sw.71 co-incubated with any of the 3 bacteria responded with significant increased secretion of interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor. All bacteria elicited the secretion of IL-6 and interferon (IFN) α2, 2 proinflammatory cytokines. In addition, Lactobacillus species resulted in increased secretion of IL-12p40 and IFNγ. While E. coli did not modify secretion of anti-inflammatory cytokines, Sw.71 cells responded to co-incubation with Lactobacillus species by secreting increased levels of IL-10 and IL-1ra. Both Lactobacillus species led to a decreased secretion of IL-4. Conclusion: All 3 bacterial species triggered significant release of chemokines and inflammatory cytokines, suggesting that a commensal relationship with trophoblasts may not be feasible.

We aimed at assessing the evidence for an effect on vaginal dysbiosis by oral administration of a mixture of Lactobacillus strains isolated from vaginal microbiota. For this purpose, we systematically reviewed the literature for randomised clinical trials (RCTs) in which the effect of oral administration of a mixture of four Lactobacillus strains (Lactobacillus crispatus LbV 88 (DSM 22566), Lactobacillus gasseri LbV 150N (DSM 22583), Lactobacillus jensenii LbV 116 (DSM 22567) and Lactobacillus rhamnosus LbV96 (DSM 22560)) on vaginal dysbiosis was examined based on Nugent score. Four RCTs were identified: a double-blind (DB)-RCT in 60 male-to-female transsexual women with neovagina; an open label RCT in 60 pregnant women with herpes virus infection; a DB-RCT in 36 women with bacterial vaginosis; a DB-RCT in 22 postmenopausal breast cancer patients receiving chemotherapy. Only in the three DB-RCTs Nugent score was assessed. The meta-analysis of these trials showed a significant reduction of Nugent score by probiotics compared to placebo in the fixed (standardised mean differences (SMD) -0.561; confidence interval (CI) -0.935 to -0.186; P=0.004 and random effect models (SMD -0.561; CI -0.935 to -0.186; P=0.004). The odds ratio (OR) of the cases presenting with improved Nugent score after probiotics compared to placebo treatment showed a significant effect in the fixed (OR=3.936; CI 1.702 to 9.100; P=0.001) and random effect model (OR=3.902; CI 1.681 to 9.059; P=0.001) Cochran’s Q and I2statistics showed no heterogeneity. This meta-analysis indicates that the oral intake of the pertinent Lactobacillus strains improves the microbial pattern in vaginal dysbiosis.

Persistent infection with High Risk-Human Papilloma Viruses (HR-HPVs) is a primary cause of cervical cancer worldwide. Vaginal-dysbiosis-associated bacteria were correlated with the persistence of HR-HPVs infection and with increased cancer risk. We obtained strains of the most represented bacterial species in vaginal microbiota and evaluated their effects on the survival of cervical epithelial cells and immune homeostasis. The contribution of each species to supporting the antiviral response was also studied. Epithelial cell viability was affected by culture supernatants of most vaginal-dysbiosis bacteria, whereas Lactobacillus gasseri or Lactobacillus jensenii resulted in the best stimulus to induce interferon-γ (IFN-γ) production by human mononuclear cells from peripheral blood (PBMCs). Although vaginal-dysbiosis-associated bacteria induced the IFN-γ production, they were also optimal stimuli to interleukin-17 (IL-17) production. A positive correlation between IL-17 and IFN-γ secretion was observed in cultures of PBMCs with all vaginal-dysbiosis-associated bacteria suggesting that the adaptive immune response induced by these strains is not dominated by TH1 differentiation with reduced availability of IFN-γ, cytokine most effective in supporting virus clearance. Based on these results, we suggest that a vaginal microbiota dominated by lactobacilli, especially by L. gasseri or L. jensenii, may be able to assist immune cells with clearing HPV infection, bypasses the viral escape and restores immune homeostasis.

Background This study aimed to investigate the specific vaginal microbiome in the differential diagnosis of endometriosis/adenomyosis (EM/AM)-associated chronic pelvic pain (CPP) from other types of CPP, and to explore the role of the vaginal microbiome in the mechanism of EM/AM-associated CPP. Methods We recruited 37 women with EM/AM-associated CPP, 25 women with chronic pelvic pain syndrome (CPPS) without EM/AM, and 66 women without CPPS into our study. All of the participants were free from human papillomavirus (HPV) infection. Sequencing of barcoded 16S rRNA gene fragments (V4) was used to determine the vaginal microbiome composition on the Illumina HiSeq2500 System. Taxonomic and functional bioinformatics analyses were performed using t-test, linear discriminant analysis effect size (LEfSe), MetaStat, and PICRUSt algorithms. Results At the species level, EM/AM-associated CPP was found to be associated with a predominance of Clostridium butyricum, Clostridium disporicum, Alloscardovia omnicolens, and Veillonella montpellierensis, and a concomitant paucity of Lactobacillus jensenii, Lactobacillus reuteri, and Lactobacillus iners. When the relative abundance of Clostridium disporicum was over 0.001105% and that of Lactobacillus reuteri was under 0.1911349%, the differential diagnostic sensitivity and specificity were 81.08% and 52.0%, respectively. When serum CA125 was combined, the sensitivity increased to 89.19%, but the specificity remained at 52.0%. The PICRUSt results identified 7 differentially regulated pathways within the 3 groups that may be of relevance. Conclusions Compared to that of CPPS patients without EM/AM and women without CPPS, the vaginal microbiome of patients with EM/AM-associated CPP shows significantly higher alpha (phylogenetic) diversity, as well as higher counts of Clostridium butyricum, Clostridium disporicum, Alloscardovia omnicolens, and Veillonella montpellierensis. These differences in the vaginal microbiome may interfere with local functional pathways, which could provide a direction for innovative metabolite-specific targeted treatment. The combination of vaginal biomarkers and serum CA125 may provide an original method to differentiate EM/AM-associated CPP.

Understanding the characteristics of the vaginal microbiota of our patients allows us to carry out both a personalized therapeutic approach and a closer follow-up in those with microbiota susceptible to dysbiosis. This trial pursues the analysis of the vaginal microbiota of premenopausal women and its fluctuations within a four-week follow-up period. Vaginal samples of 76 fertile women were taken at a baseline visit and at a final visit (day 28 ± 5). To perform a phylogenetic study, we employed massive sequencing techniques to detect the 16S rRNA gene of the vaginal microbiota. The most prevalent vaginal microbial community was type I (34.87%), dominated by Lactobacillus crispatus. Vaginal microbial community types II (Lactobacillus gasseri) and V (Lactobacillus jensenii) were underrepresented in our population. When repeating the sampling process four weeks later, 75% of our patients maintained their initial bacterial community. In the follicular phase, the most recurrent microbiota was type III (Lactobacillus iners), in the periovulatory phase, types III and IV (microbial diversity), finally, in the luteal phase, the most frequent type was IV. The most prevalent vaginal bacterial community in our population was dominated by L. crispatus. The vaginal microbiota was resistant to changes in its bacterial community in 75% of our patients, even between consecutive menstrual cycles.

The vaginal microbiome plays an important role in human health and species of vaginal bacteria have been associated with reproductive disease. Strain-level variation is also thought to be important, but the diversity, structure and evolutionary history of vaginal strains is not as well characterized. We developed and validated an approach to measure strain variation from metagenomic data based on SNPs within the core genomes for six species of vaginal bacteria: Gardnerella vaginalis , Lactobacillus crispatus , Lactobacillus iners , Lactobacillus jensenii , Lactobacillus gasseri and Atopobium vaginae . Despite inhabiting the same environment, strain diversity and structure varies across species. All species except L. iners are characterized by multiple distinct groups of strains. Even so, strain diversity is lower in the Lactobacillus species, consistent with a more recent colonization of the human vaginal microbiome. Both strain diversity and the frequency of multi-strain samples is related to species-level diversity of the microbiome in which they occur, suggesting similar ecological factors influencing diversity within the vaginal niche. We conclude that the structure of strain-level variation provides both the motivation and means of testing whether strain-level differences contribute to the function and health consequences of the vaginal microbiome.

Immunobiotics have emerged as a promising intervention to alleviate intestinal damage in inflammatory bowel disease (IBD). However, the beneficial properties of immunobiotics are strain dependent and, therefore, each strain has to be evaluated in order to demonstrate its potential application in IBD. Our previous in vitro and in vivo studies demonstrated that Lactobacillus jensenii TL2937 attenuates gut acute inflammatory response triggered by Toll-like receptor 4 activation. However, its effect on colitis has not been evaluated before. In this work, we studied whether the TL2937 strain was able to protect against the development of colitis in a dextran sodium sulfate (DSS)-induced mouse model and we delved into the mechanisms of action by evaluating the effect of the immunobiotic bacteria on the transcriptomic response of DSS-challenged intestinal epithelial cells. L. jensenii TL2937 was administered to adult BALB/c mice before the induction of colitis by the administration of DSS. Colitis and the associated inflammatory response were evaluated for 14 days. Mice fed with L. jensenii TL2937 had lower disease activity index and alterations of colon length when compared to control mice. Reduced myeloperoxidase activity, lower production of pro-inflammatory (TNF-α, IL-1, CXCL1, MCP-1, IL-15, and IL-17), and higher levels of immunoregulatory (IL-10 and IL-27) cytokines were found in the colon of TL2937-treated mice. In addition, the treatment of porcine intestinal epithelial (PIE) cells with L. jensenii TL2937 before the challenge with DSS differentially regulated the activation of the JNK pathway, leading to an increase in epithelial cell integrity and to a differential immunotranscriptomic response. TL2937-treated PIE cells had a significant reduction in the expression of inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6, IL-15), chemokines (CCL2, CCL4, CCL8, CXCL4, CXCL5, CXCL9, CXCL10), adhesion molecules (SELE, SELL, EPCAM), and other immune factors (NCF1, NCF2, NOS2, SAA2) when compared to control cells after the challenge with DSS. The findings of this work indicate that (a) L. jensenii TL2937 is able to alleviate DSS-induced colitis suggesting a potential novel application for this immunobiotic strain, (b) the modulation of the transcriptomic response of intestinal epithelial cells would play a key role in the beneficial effects of the TL2937 strain on colitis, and (c) the in vitro PIE cell immunoassay system could be of value for the screening and selection of new immunobiotic strains for their application in IBD. Fil: Sato, Nana. Tohoku University; Japón Fil: García Castillo, Valeria. Tohoku University; Japón. Universidad de Concepción; Chile Fil: Yuzawa, Mao. Tohoku University; Japón Fil: Islam, Md Aminul. Tohoku University; Japón. Bangladesh Agricultural University; Bangladesh Fil: Albarracín, Leonardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentina. Tohoku University; Japón Fil: Tomokiyo, Mikado. Tohoku University; Japón Fil: Ikeda Ohtsubo, Wakako. Tohoku University; Japón Fil: García Cancino, Apolinaria. Universidad de Concepción; Chile Fil: Takahashi, Hideki. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Kitazawa, Haruki. Tohoku University; Japón

Lactobacillus jensenii is an anaerobic bacterium found in the urogenital tract that is known to prevent common vaginal infections. Recently, it was divided into two species, L. jensenii and L. mulieris . Here, we report the draft genome sequence of L. mulieris UMB9245, with a genome length of 1,723,383 bp assembled into 52 contigs.

ObjectivesVaginal washing has been associated with reductions in cultivable Lactobacillus and an increased risk of both bacterial vaginosis (BV) and HIV infection. The effect of vaginal washing on the quantity of individual Lactobacillus species is not well characterised. This analysis tested the hypothesis that vaginal washing would be associated with a lower likelihood of Lactobacillus spp. detected by both culture and quantitative PCR (qPCR).MethodsWe conducted a cross-sectional study of 272 HIV-seronegative women enrolled in an open-cohort study in Mombasa, Kenya. Vaginal washing and sexual risk behaviours were assessed using face-to-face interviews. Vaginal Lactobacillus spp. were detected using cultivation and PCR methods, with L. crispatus, L. jensenii and L. iners concentrations measured using qPCR assays targeting the 16S rRNA gene. Poisson regression with robust SEs was used to assess associations between vaginal washing and Lactobacillus detection by culture and qPCR.ResultsEighty percent (n=217) of participants reported vaginal washing in the prior week. One-fifth (n=58) of participants had BV by Nugent score. In unadjusted analysis, vaginal washing was associated with a 45% decreased likelihood of Lactobacillus spp. detection by culture (prevalence ratio (PR): 0.55, 95% CI 0.37 to 0.82). Adjusting for age and condomless sex in the prior week did not change the magnitude of the association (adjusted PR (aPR): 0.56, 95% CI (0.37 to 0.85). Vaginal washing was associated with approximately a 40% reduction in L. crispatus detection (aPR: 0.57, 95% CI 0.36 to 0.92), but was not significantly associated with L. jensenii (aPR: 0.68, 95% CI 0.42 to 1.09) or L. iners detection (aPR: 1.03, 95% CI 0.92 to 1.15).ConclusionsVaginal washing in the prior week was associated with a significantly reduced likelihood of detecting cultivable Lactobacillus and L. crispatus by qPCR. Given associations between Lactobacillus detection and improved reproductive health outcomes, these results provide motivation for additional study of vaginal washing cessation interventions to improve vaginal health.

Background The bacterial community present in the female lower genital tract plays an important role in maternal and neonatal health. Imbalances in this microbiota have been associated with negative reproductive outcomes, such as spontaneous preterm birth (sPTB), but the mechanisms underlying the association between a disturbed microbiota and sPTB remain poorly understood. An intrauterine infection ascending from the vagina is thought to be an important contributor to the onset of preterm labour. Our objective was to characterize the vaginal microbiota of pregnant women who had sPTB (n = 46) and compare to those of pregnant women who delivered at term (n = 170). Vaginal swabs were collected from women at 11–16 weeks of gestational age. Microbiota profiles were created by PCR amplification and pyrosequencing of the cpn60 universal target region. Results Profiles clustered into seven community state types: I (Lactobacillus crispatus dominated), II (Lactobacillus gasseri dominated), III (Lactobacillus iners dominated), IVA (Gardnerella vaginalis subgroup B or mix of species), IVC (G. vaginalis subgroup A dominated), IVD (G. vaginalis subgroup C dominated) and V (Lactobacillus jensenii dominated). The microbiota of women who experienced preterm birth (

An issue that is currently undergoing extensive study is the influence of human vaginal microbiota (VMB) on the health status of women and their neonates. Healthy women are mainly colonised with lactobacilli such as Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners; however, other bacteria may be elements of the VMB, particularly in women with bacterial vaginosis. The implementation of culture-independent molecular methods in VMB characterisation, especially next-generation sequencing, have provided new information regarding bacterial diversity in the vagina, revealing a large number of novel, fastidious, and/or uncultivated bacterial species. These molecular studies have contributed new insights regarding the role of bacterial community composition. In this study, we discuss recent findings regarding the reproductive tract microbiome. Not only bacteria but also viruses and fungi constitute important components of the reproductive tract microbiome. We focus on aspects related to the impact of the maternal microbiome on foetal development, as well as the establishment of the neonatal microbiomes, including the placenta microbiome, and the haematogenous source of intrauterine infection. We also discuss whether the role of the vaginal microbiome is currently understood and appreciated.

Introduction. In healthy women of reproductive age, the vaginal microflora is represented mainly by lactobacilli. They provide a barrier function, preventing the propagation of opportunistic pathogens and the colonization of the vagina by pathogenic microorganisms. It is shown that the lactobacillary microflora of the vagina is very diverse, but not all species of lactobacilli can provide reliable protection of the female organism. Objective: to characterize the species and quantitative composition of vaginal lactobacilli of women of reproductive age in norm and in dysbiosis. Material and methods. The study involved 123 patients from polyclinic departments of the D.O. Ott Reasearch Institute of Obstetrics, Gynecology and Reproductology. For analysis of clinical material (vaginal discharge) for lactobacilli and other microorganisms, quantitative real-time PCR was used. Results. The most common types of lactobacilli are Lactobacillus jensenii, L. Iners, L. crispatus, L. vaginalis and L. gasseri. Species diversity of lactobacilli (detection of ≥2 species) is observed much more often in women with physiological microbiocenosis than in women with vaginal dysbiosis. Our study confirms that L. crispatus is the dominant species of the vaginal biotope of healthy women, while in dysbiosis the species L. gasseri and L. Iners are most often identified.

PURPOSE: To date, molecular methods that circumvent the limitations of traditional culture methods have not been used to describe the vaginal microflora in India. Here, we compared culture and culture-independent molecular methods in characterizing the vaginal microbiota in Indian women. METHODOLOGY: Culture methods involved traditional cultivation on Rogosa and sheep blood agar, whereas culture-independent methods bypassed a culturing step by performing broadrange 16S rDNA PCR on DNA isolated directly from vaginal swabs. RESULTS: A total of 13 women were included in the study, of which five were characterized as healthy, two were bacterial vaginosis intermediate and six were bacterial vaginosis positive according to Nugent scoring. Lactobacillus jensenii was detected most frequently when using culture methods. On the other hand, Lactobacillus iners, which was not detected by culture methods, was the most common Lactobacillus sp. detected using cultivation-independent methods. CONCLUSION: We found little overlap between the species found using cultivation-dependent and cultivation-independent methods. Rather, culture-dependent and culture-independent methods were found to be complementary in describing the vaginal microflora among South Indian women. Culture-independent methods were found to be superior in detecting clinically relevant vaginal flora.

Objectives Lactobacillus probiotics have been proposed as an antibiotic-sparing prevention strategy for urinary tract infections (UTIs). Our objective was to examine the relative ability of the 4 most common vaginal Lactobacillus species to inhibit the growth of Escherichia coli, the most common cause of UTIs. Methods Conditioned media (CM) was created from 4 laboratory strains of Lactobacillus species: Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners, and 6 clinical strains of L. crispatus. One laboratory strain of E. coli was cocultured with each CM, as well as with various acidic solutions. Three clinical strains of E. coli from women with acute cystitis were cocultured with the CM from a laboratory strain of L. crispatus. Bacterial growth was compared between experimental variants and media control using analysis of variance. Results Growth of E. coli was inhibited by CM from L. crispatus, L. jensenii, and L. gasseri, but not L. iners. The magnitude of inhibition was correlated with the pH of the individual CM and the concentration of D-lactic acid. Different acids inhibited E. coli growth in proportion to the pH of the acid solution. Similar levels of inhibition were seen when L. crispatus was incubated with clinical E. coli strains as with laboratory E. coli. Conclusions Three of the most common vaginal Lactobacillus species inhibit E. coli growth, likely through creating a low pH environment. However, L. iners, one of the most common species found after menopause, does not. These findings might be leveraged to more effectively manage UTIs.

The physiological microbiota of the vagina is responsible for providing a protective barrier, but Some factors can disturb the balance in its composition. At that time, the amounts of the genus Lactobacillus decrease, which may lead to the development of infection and severe complications during pregnancy. The aim of the study was the analysis of the bacterial composition of the vagina in 32 Caucasian women at each trimester of pregnancy using the next-generation sequencing method and primers targeting V3-V4 regions. In the studied group, the dominant species were Lactobacillus iners, Lactobacillus gasseri, and Lactobacillusplantarum. Statistically significant differences in the quantitative composition between trimesters were observed in relation to Lactobacillus jensenii,Streptococcus agalactiae, Lactobacillus iners, Gardnerella spp. Out of the 32 patients, 20 demonstrated fluctuations within the genus Lactobacillus, and 9 of them, at different stages of pregnancy, exhibited the presence of potentially pathogenic microbiota, among others: Streptococcus agalactiae, Gardnerella spp., Atopobium vaginae, and Enterococcus faecalis. The composition of the vaginal microbiota during pregnancy was subject to partial changes over trimesters. Although in one-third of the studied patients, both the qualitative and quantitative composition of microbiota was relatively constant, in the remaining patients, physiological and potentially pathogenic fluctuations were distinguished.

Lactobacillus jensenii, a protective bacterium in the vaginal microbiota, is also a member of the female urinary tract community. Here, we report 11 genome sequences of L. jensenii strains isolated from catheterized urine from women. This effort greatly increases our knowledge of the genetic diversity of this species within the bladder.

BackgroundPreterm birth is one of the leading causes of neonatal mortality. The causes for spontaneous preterm birth (PTB) are multifactorial and remain often unknown. In this study, we tested the hypothesis that human milk oligosaccharides (HMOs) in blood and urine modulate the maternal urinary and vaginal microbiome and influence the risk for PTB. We analyzed the vaginal and urinary microbiome of a cross-sectional cohort of women with and without preterm labor and correlated our findings with measurements of metabolites and HMOs in urine and blood.ResultsWe identified several microbial signatures associated with short cervix, PTB and/or preterm contractions such asLactobacillus jensenii,L. gasseri,Ureaplasma sp. andGardnerella sp..Additionally, we observed associations between sialylated HMOs, in particular 3’-sialyllactose, with PTB, short cervix and increased inflammation and confirmed an influence of HMOs on the microbiome profile.ConclusionsIdentifying serum and urinary HMOs and several key microorganisms associated with PTB, our findings point at two distinct processes modulating the risk for PTB. One process seems to be driven by sterile inflammation, characterized by increased concentrations of sialylated HMOs in serum. Another process might be microbiome-mediated, potentially driven by secretor-active HMOs in urine. Our results support current efforts to improve diagnostics and therapeutic strategies.

IgA-coated Lactobacillus live in the mucous layer of the human or mammalian intestine in close proximity to epithelial cells. They act as potential probiotics for functional food development, but their physiological regulation has not yet been studied. We isolated IgA-targeted (Lactobacillus jensenii IgA21) and lumen lactic acid bacterial strains (Pediococcus acidilactici FS1) from the fecal microbiota of a healthy woman. C57BL/6 mice were fed a normal (CON) or high fat diet (HFD) for 6 weeks and then treated with IgA21 or FS1 for 4 weeks. HFD caused dyslipidemia, mucosal barrier damage, and intestinal microbiota abnormalities. Only IgA21 significantly inhibited dyslipidemia and gut barrier damage. This was related to significant up-regulation of mucin-2, PIgR mRNA expression, and colonic butyrate production (P < 0.05 vs. HFD). Unlike IgA21, FS1 caused a more pronounced gut dybiosis than did HFD, and, in particular, it induced a significant decrease in the Bacteroidales S24-7 group and an increase in Desulfovibrionaceae (P < 0.05 vs. CON). In conclusion, IgA-coated and non-coated lactic acid bacteria of gut have been demonstrated to differentially affect the intestinal barrier and serum lipids. This indicates that IgA-bound bacteria possess the potential to more easily interact with the host gut to regulate homeostasis.

Bacterial vaginosis (BV) is the most common cause of abnormal vaginal discharge for women of childbearing age. Despite many confirming surveys, which proved the relationship between BV and human papillomavirus (HPV) infection, but this remains a matter of debate. The objective of this original article is to clarify BV is associated with HPV infection in Iranian women by quantitative real-time polymerase chain reaction (qPCR) method. The vaginal swabs of seventy-five women (55 and 20 samples from patients and controls, respectively) with and without manifestation of HPV were collected during February 2018 and December 2018, and extracted DNA was assayed by real-time PCR looking for the HPV serotypes and bacterial load by using bacterial 16S rRNA gene. A total of 55 cervical, 13 (23.4%) isolates were HPV-negative, and 42 (76.4%) isolates were HPV-positive. In total, 83.3% of HPV-positive isolates had BV infection. The hotspot of incidence with BV is between the ages of 26–34 years. Although, we can see there are low frequency of Lactobacillus spp. in HPV 16 patients among other groups but unfortunately, we can't approve it with significant value (P-value >0.05). Lactobacillus gasseri and Lactobacillus jensenii showed higher colony count in BV women that infected with Gardnerella vaginalis than healthy women. The number of L. gasseri and L. rhamnosus are directly related to reducing the risk of HPV infection (P-value

Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco’s Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract.

Microorganisms with the ability to modulate the immune system (immunobiotics) have shown to interact with different pattern recognition receptors (PRRs) expressed in nonimmune and immune cells and exert beneficial effects on host's health maintenance and promotion. Suitable assay systems are necessary for an efficient and rapid screening of potential immunobiotic strains. More than a decade of research have allowed us to develop efficient in vitro models based on porcine receptors and cells (porcine immunoassay systems) to study the immunomodulatory effects of lactic acid bacteria (LAB). In addition, detailed studies of model immunobiotic LAB strains with proved abilities to improve immune health in humans (Lactobacillus rhamnosus CRL1505) or pigs (Lactobacillus jensenii TL2937) allowed us to select the most suitable biomarkers that have to be evaluated in those porcine immunoassay systems. Our in vitro models based on transfectant cells expressing porcine PRRs as well as an originally established porcine intestinal epitheliocyte (PIE) cell line have shown to be useful in vitro tools for the selection of immunobiotics and for obtaining information to elucidate the molecular mechanisms behind their beneficial effects.

Perturbations to the vaginal microbiota can lead to dysbiosis, including bacterial vaginosis (BV), which affects a large portion of the female population. In a healthy state, the vaginal microbiota is characterized by low diversity and colonization by Lactobacillus spp., whereas in BV, these species are displaced by a highly diverse population of bacteria associated with adverse vaginal health outcomes. Since prebiotic ingestion has been a highly effective approach to invigorate lactobacilli for improved intestinal health, we hypothesized that these compounds could stimulate lactobacilli at the expense of BV organisms to maintain vaginal health. Monocultures of commensal Lactobacillus crispatus , Lactobacillus vaginalis , Lactobacillus gasseri , Lactobacillus johnsonii , Lactobacillus jensenii , and Lactobacillus iners , in addition to BV-associated organisms and Candida albicans , were tested for their ability to utilize a representative group of prebiotics consisting of lactitol, lactulose, raffinose, and oligofructose. The disaccharide lactulose was found to most broadly and specifically stimulate vaginal lactobacilli, including the strongly health-associated species L. crispatus , and importantly, not to stimulate BV organisms or C. albicans . Using freshly collected vaginal samples, we showed that exposure to lactulose promoted commensal Lactobacillus growth and dominance and resulted in healthy acidity partially through lactic acid production. This provides support for further testing of lactulose to prevent dysbiosis and potentially to reduce the need for antimicrobial agents in managing vaginal health. IMPORTANCE Bacterial vaginosis (BV) and other dysbioses of the vaginal microbiota significantly affect the quality of life of millions of women. Antimicrobial therapy is often poorly effective, causes side effects, and does not prevent recurrences. We report one of very few studies that have evaluated how prebiotics—compounds that are selectively fermented by beneficial bacteria such as Lactobacillus spp.—can modulate the vaginal microbiota. We also report use of a novel in vitro polymicrobial model to study the impact of prebiotics on the vaginal microbiota. The identification of prebiotic lactulose as enhancing Lactobacillus growth but not that of BV organisms or Candida albicans has direct application for retention of homeostasis and prevention of vaginal dysbiosis and infection.

BackgroundBacterial vaginosis (BV) is a microbial imbalance (i.e., dysbiosis) that can produce serious medical effects in women at childbearing age. Little is known, however, about the incidence of BV or vaginal microbiota dysbiosis in pregnant teenagers in low and middle-income countries such as Ecuador. The scope of this exploratory analysis was to study the relationship between epidemiologic and microbial risk factors. Among the microbiology risk factors this study investigated fiveLactobacillusspecies, two of them know in preview studies as microbiology risk factors for BV development (Lactobacillus acidophilusandLactobacillus iners), and the last three known for being associated with a healthy vaginal tract (Lactobacillus crispatus,Lactobacillus gasseriandLactobacillus jensenii). In addition, fastidious anaerobes known to be microbial risk factors for BV development in pregnant teenagers were searched as well, more exactly,Gardnerella vaginalis,Atopobium vaginaeandMobiluncus mulieris.MethodsNinety-five healthy adolescent pregnant women, visiting a secondary level hospital in Quito, Ecuador, were enrolled into the study in 2015. The enrolled patients were between 10 to 13 weeks of pregnancy. Four epidemiological risk factors were collected in a survey: age, civil status, sexual partners and condom use. Also, vaginal pH was measured as a health risk factor. DNA was extracted from endocervical and exocervical epithelia from all the patients’ samples. PCR analysis was performed in order to characterize the presence of the eight bacterial species known as risk factors for BV development, targeting three anaerobes and fiveLactobacillusspecies. Univariate and multivariate analysis were performed to identify associated factors for the presence of anaerobic species using logistic regression.ResultsThe 95 vaginal microflora samples of these teenagers were analyzed. Two of the bacterial species known to cause BV:A. vaginae(100%) andG. vaginalis(93.7%) were found in high prevalence. Moreover, the most predominant bacterialLactobacillusspecies found in the pregnant teenagers’ vaginal tract wereL. crispatus(92.6%),L. iners(89.5%) andL. acidophilus(87.4%). In addition, the average vaginal pH measured in the study population was 5.2, and high pH was associated with the presence of the three-anaerobic species (p = 0.001). Finally,L. jensenii’s presence in the study decreased in 72% the occupation of the three anaerobes.DiscussionThis work identified a high pH as a risk factor for BV anaerobes’ presence in adolescent pregnant women. Moreover, this study identifiedL. crispatus, L. inersandL. acidophilusto be the most abundant species in our study population. From all fastidious anaerobes analyzed in this study,A. vaginaewas present in all pregnant teenagers. To conclude,L. jenseniicould be a potential healthy vaginal microbiota candidate in pregnant teenagers and should be further analyzed in future studies.

Today there is an urgent need for safe and effective dual-purpose contraceptive agents, which can simultaneously prevent unintended pregnancies and sexually transmitted infections (STI). There are several naturally occurring antimicrobial and antibiotic drugs (novobiocin, coumermycin and chlorobiocin) reported in the literature, which are based on 4-hydroxy coumarins as the active pharmacophore. Based on these interesting reports, we designed and synthesized a library of new 4-hydroxy coumarin derivatives and evaluated them for spermicidal activity. Among the tested compounds, two compounds (2a and 2d) displayed better activity (greater than 95% sperm immobilization at 0.5 mM concentration) than the positive control nonoxynol-9 (N-9). Furthermore, all the compounds were screened for antimicrobial activity against different strains of Trichomonas vaginalis and two compounds (2c and 2h) exhibited potent activity as compared to the reference drug metronidazole. The cytotoxicity assay showed that most of these compounds were safer than the N-9 against the human cervical HeLa cell line and normal vaginal flora Lactobacillus jensenii strains. The studies have demonstrated that compound (2a) is a potential lead to develop a dually active vaginal contraceptive.

Live biotherapeutic products (LBPs), commonly referred to as probiotics, are typically preparations of live bacteria, such as Lactobacillus and Bifidobacterium species that are considered normal human commensals. Popular interest in probiotics has been increasing with general health benefits being attributed to their consumption, but there is also growing interest in evaluating such products for treatment of specific diseases. While over-the-counter probiotics are generally viewed as very safe, at least in healthy individuals, it must be remembered that clinical studies to assess these products may be done in individuals whose defenses are compromised, such as through a disease process, immunosuppressive clinical treatment, or an immature or aging immune system. One of the major safety criteria for LBPs used in clinical studies is microbial purity, i.e., the absence of extraneous, undesirable microorganisms. The main goal of this project is to develop recombinant phage lysins as reagents for improved purity assays for LBPs. Phage lysins are hydrolytic enzymes containing a cell binding domain that provides specificity and a catalytic domain responsible for lysis and killing. Our approach is to use recombinant phage lysins to selectively kill target product bacteria, which when used for purity assays will allow for outgrowth of potential contaminants under non-selective conditions, thus allowing an unbiased assessment of the presence of contaminants. To develop our approach, we used LysA2, a phage lysin with reported activity against a broad range of Lactobacillus species. We report the lytic profile of a non-tagged recombinant LysA2 against Lactobacillus strains in our collection. We also present a proof-of-concept experiment, showing that addition of partially purified LysA2 to a culture of Lactobacillus jensenii (L. jensenii) spiked with low numbers of Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus ) effectively eliminates or knocks down L. jensenii, allowing for clear detection of the contaminating strains. With continued identification and characterization of phage lysins, we hope that the use of recombinant phage lysins in purity assays for products containing live microbials may offer additional tools to help advance product development of LBPs.

Background Cervicitis is an inflammatory condition of the cervix associated with upper genital tract infection and reproductive complications. Although cervicitis can be caused by several known pathogens, the etiology frequently remains obscure. Here we investigate vaginal bacteria associated with bacterial vaginosis as potential causes of cervicitis. Methods Associations between vaginal bacteria and cervicitis were assessed in a retrospective case-control study of women attending a Seattle sexually transmitted disease clinic. Individual bacterial species were detected using 2 molecular methods: quantitative polymerase chain reaction (qPCR) and broad-range 16S rRNA gene PCR with pyrosequencing. The primary finding from this initial study was evaluated using qPCR in a second cohort of Kenyan women. Results The presence of Mageeibacillus indolicus, formerly BVAB3, in the cervix was associated with cervicitis, whereas the presence of Lactobacillus jensenii was inversely associated. Quantities of these bacteria did not differ between cervicitis cases and controls, although in a model inclusive of presence and abundance, M. indolicus remained significantly associated with cervicitis after adjustment for other cervicitis-causing pathogens. M. indolicus was not associated with cervicitis in our study of Kenyan women, possibly due to differences in the clinical definition of cervicitis. Conclusions Colonization of the endocervix with M. indolicus may contribute to the clinical manifestations of cervicitis, but further study is needed to determine whether this finding is repeatable and applicable to diverse groups of women. Colonization of the cervix with L. jensenii could be a marker of health, perhaps reducing inflammation or inhibiting pathogenic infection.

Bacterial vaginosis (BV) is characterised by a depletion of lactobacilli in favour of an overgrowth of anaerobic bacteria. It is associated with increased risk for urogenital infections and abortion. In this study we assessed the effect of a yoghurt drink containing Lactobacillus strains on BV. The strains had been isolated from healthy pregnant women and selected for acidification capacity, production of H2O2, glycogen utilisation, bile salt tolerance and inhibition of pathogens. Using Amsel criteria BV was diagnosed in 36 women aged ≥18 years with stable menstrual cycle or menopause. They were treated with oral metronidazole for 7 days (2×500 mg/d). Starting with the treatment, women consumed twice daily either verum or placebo during 4 weeks. Verum was 125 g yoghurt containing (besides Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) living strains Lactobacillus crispatus LbV 88 (DSM 22566), Lactobacillus gasseri LbV 150N (DSM 22583), Lactobacillus jensenii LbV 116 (DSM 22567) and Lactobacillus rhamnosus LbV96 (DSM 22560), each 1×107 cfu/ml; placebo was 125 g chemically acidified milk. After 4 weeks of intervention 0 of 17 had BV in the verum group versus 6 of 17 in the s.a. control (0.018 in Fisher Exact test). Amsel score decreased during the intervention period by 4.0 (median) (4.0; 3.0) (25th; 75th percentile) in the verum group compared to 2.0 (4.0; 0.0) in the control group (P=0.038 in Mann-Whitney test). Discharge and odour (Amsel criteria 2+3) also decreased by 2.0 (2.0; 1.0) in the verum compared to 1.0 (2.0; 0.0) in the control group (P=0.01) and differed after 4 weeks intervention between the groups 0.0 (0.0; 0.0) versus 1.0 (0.0; 2.0) (P=0.001). Nugent score decreased during the intervention period by 5.5 (7.0;2.3) in the verum compared to 3.0 (6.0;0.5) in the control group (P=0.158). Additional intake of yoghurt containing these probiotic strains improved the recovery rate and symptoms of BV and tended to improve the vaginal microbial pattern.

Introduction Incident bacterial vaginosis (BV) is associated with a shift in the vaginal microbiota from lactobacillus predominant to a diverse community of anaerobic bacteria The kinetics of this shift are poorly described. We sought to identify the dynamics of incident BV. Methods Twenty women with frequent BV (>3 episodes per year) self-collected vaginal swabs every 8 hours for 60 days. Swabs were analysed with quantitative PCR targeting Lactobacillus iners, Lactobacillus jensenii, Lactobacillus crispatus, Gardenerella vaginalis, Atopobium vaginae, bacterial vaginosis associated bacterium-2 (BVAB2), and Megasphaera spp . We defined bacteria as absent, low (4 DNA copies), moderate (104–108 DNA copies) or high-level (108–1011 DNA copies). Participants kept detailed diaries regarding sexual behaviour, menstruation, antibiotic use and vulvovaginal symptoms. Results We noted three states of the vaginal microbiota: three women had high-level lactobacilli with intermittent, transient low-level G. vaginalis throughout the sampling period (State 1); five women had high-level lactobacilli with persistent, fluctuating low to moderate-level G. vaginalis and other BV associated anaerobic species throughout the sampling period (State 2); two women had polymicrobial colonisation with high-level G. vaginalis and other BV associated species, and intermittent, transient low-level, or persistent moderate-level Lactobacillus jensenii and crispatus throughout the sampling period (State 3). Ten women shifted between States 2 and 3 on at least one occasion. Extremely rapid transition from State 2 to 3 over. Conclusions The vaginal microbiota is extremely dynamic and BV develops over narrow time intervals. Low-levels of BV associated species in the vagina may be a risk factor for rapid, incident BV. Future studies will identify drivers of shifts in the vaginal bacterial biota.

Lactobacillus jensenii SNUV360 is a potential probiotic strain that shows antimicrobial activity for the treatment of bacterial vaginosis. Here, we present the complete genomic sequence of L. jensenii SNUV360, isolated from a vaginal sample from a healthy Korean woman. Analysis of the sequence may provide insight into its functional activity.

The demand on thermostable d -lactate dehydrogenases ( d -LDH) has been increased for d -lactic acid production but thermostable d -DLHs with industrially applicable activity were not much explored. To identify a thermostable d -LDH, three d -LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli . One of the three d -LDHs ( d -LDH3) exhibited higher optimal reaction temperature (50 °C) than the others. The T 50 10 value of this thermostable d -LDH3 was 48.3 °C, much higher than the T 50 10 values of the others (42.7 and 42.9 °C) and that of a commercial d -lactate dehydrogenase (41.2 °C). The T m values were 48.6, 45.7 and 55.7 °C for the three d -LDHs, respectively. In addition, kinetic parameter ( k cat / K m ) of d -LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 °C, implying that d -lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the d -LDH3 characterized in this study a good candidate for the microbial production of d -lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial.

In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable Enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs. Fil: Kobayashi, Hisakazu. Tohoku University; Japón Fil: Albarracín, Leonardo Miguel. Tohoku University; Japón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sato, Nana. Tohoku University; Japón Fil: Kanmani, Paulraj. Tohoku University; Japón Fil: Kober, Humayun A.K.M.. Tohoku University; Japón. Chittagong Veterinary and Animal Sciences University. Department of Dairy and Poultry Science; Bangladesh Fil: Ikeda-Ohtsubo, Wakako. Tohoku University; Japón Fil: Suda, Yoshihito. Miyagi University; Japón Fil: Nochi, Tomonori. Tohoku University; Japón Fil: Aso, Hisashi. Tohoku University; Japón Fil: Makino, Seiya. Meiji Co., Ltd. Food Science Research Labs.; Japón Fil: Kano, Hiroshi. Meiji Co., Ltd. Food Science Research Labs.; Japón Fil: Ohkawara, Sou. Meiji Seika Pharma Co., Ltd. Agricultural and Veterinary Division; Japón Fil: Saito, Tadao. Tohoku University; Japón Fil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Tohoku University; Japón Fil: Kitazawa, Haruki. Tohoku University; Japón

BACKGROUND Bacterial vaginosis (BV) is characterized by low abundance of Lactobacillus species, high pH, and immune cell infiltration and has been associated with an increased risk of human papillomavirus (HPV) infection. We molecularly assessed the cervicovaginal microbiota over time in human immunodeficiency virus (HIV)-infected and HIV-uninfected women to more comprehensively study the HPV-microbiota relationship, controlling for immune status. METHODS 16S ribosomal RNA gene amplicon pyrosequencing and HPV DNA testing were conducted annually in serial cervicovaginal lavage specimens obtained over 8-10 years from African American women from Chicago, of whom 22 were HIV uninfected, 22 were HIV infected with a stable CD4+ T-cell count of > 500 cells/mm3, and 20 were HIV infected with progressive immunosuppression. Vaginal pH was serially measured. RESULTS The relative abundances of Lactobacillus crispatus and other Lactobacillus species were inversely associated with vaginal pH (all P < .001). High (vs low) L. crispatus relative abundance was associated with decreased HPV detection (odds ratio, 0.48; 95% confidence interval, .24-.96; Ptrend = .03) after adjustment for repeated observation and multiple covariates, including pH and study group. However, there were no associations between HPV and the relative abundance of Lactobacillus species as a group, nor with Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii individually. CONCLUSIONS L. crispatus may have a beneficial effect on the burden of HPV in both HIV-infected and HIV-uninfected women (independent of pH).

Background. Bacterial vaginosis (BV) represents shifts in microbiota from Lactobacillus spp. to diverse anaerobes. Although antibiotics relieve symptoms and temporarily eradicate BV-associated bacteria (BVAB), BV usually recurs. We investigated the role of extravaginal BVAB reservoirs in recurrence. Methods. Risks for BV acquisition over the course of 1 year were defined. DNA in vaginal, anal, and oral swab samples from enrollment was subjected to quantitative polymerase chain reaction assays targeting 16S ribosomal RNA genes of Gardnerella vaginalis, Lactobacillus crispatus, BVAB1, BVAB2, BVAB3, Megasphaera spp., Lactobacillus jensenii, and Leptotrichia/Sneathia spp. A case-control approach analyzed BVAB detection at enrollment for case patients (BV acquisition) versus controls (none). Results. Of 239 women enrolled without BV, 199 were seen in follow-up, and 40 experienced BV; 15 had all samples for analysis. Detection of G. vaginalis in oral cavity or anal samples and Leptotrichia/Sneathia spp. in anal samples was more common at enrollment among case patients, who also had higher concentrations of these bacteria and Megasphaera relative to 30 controls at each site. In contrast, L. crispatus was detected more frequently in anal samples among controls. Conclusions. Women who acquire BV are more likely have previous colonization of extravaginal reservoirs with some BVAB, and less likely to have L. crispatus, suggesting that BVAB may be acquired vaginally from extravaginal reservoirs. Bacterial vaginosis (BV) is the most prevalent vaginal infection in reproductive-age women, and has been consistently associated with adverse outcomes related to upper genital tract complications and with increased risk of human immunodeficiency virus acquisition [1–3]. Of 3739 women enrolled during 2001–2004 in a nationally representative sample of the US civilian

Most human immunodeficiency virus (HIV) transmissions in women occur through the cervicovaginal mucosa, which is coated by a bacterial biofilm including Lactobacillus. This commensal bacterium has a role in maintaining a healthy mucosa and can be genetically engineered to produce antiviral peptides. Here, we report a 63% reduction in transmission of a chimeric simian/HIV (SHIV(SF162P3)) after repeated vaginal challenges of macaques treated with Lactobacillus jensenii expressing the HIV-1 entry inhibitor cyanovirin-N. Furthermore, peak viral loads in colonized macaques with breakthrough infection were reduced sixfold. Colonization and prolonged antiviral protein secretion by the genetically engineered lactobacilli did not cause any increase in proinflammatory markers. These findings lay the foundation for an accessible and durable approach to reduce heterosexual transmission of HIV in women, which is coitally independent, inexpensive, and enhances the natural protective effects of the vaginal microflora.

Lactobacillus is a rare cause of endocarditis and has been linked to structural heart disease, invasive procedures, prosthetic valves, and dental infections. We report for the first time a case of Lactobacillus endocarditis that occurred in a 31-year-old woman without a medical history 1 month after her first pregnancy and vaginal delivery involving not only the native aortic valve but also the posterior part of ascending aorta, the roof of the left atrium, and the intra-atrial septum, requiring a complex surgical procedure.

Lactobacilli have been shown to inhibit in vitro growth of many pathogens and have been used as probiotics to treat a broad range of gastrointestinal and/or vaginal disorders. We sought to determine the in vitro inhibitory potential of lactobacilli of vaginal origin to some bacteria associated with bacterial vaginosis (BV), to characterize the inhibitory substances produced by these lactobacilli and to assess H2O2 production. Vaginal specimens were obtained by swabbing the lateral vaginal walls from 107 women two months following BV treatment. One hundred and fifty eight Lactobacillus spp. were isolated in 82 of the 107 women. Lactobacillus jensenii was the predominant strain isolated among these women (29/158; 18.4%). Among 158 culture supernatants tested for antibacterial activity against BV-associated bacteria, none inhibited the growth of Bacteroides fragilis while 23% (37/158), 28% (45/158) and 29% (46/158) inhibited the growth of Prevotella bivia, Gardnerella vaginalis and Mobiluncus spp. respectively. The lactobacilli produced supernatants with a pH range between 2.62 and 6.71; the highly acidic (pH 2-3.99) supernatants were more inhibitory to the indicator strains. There was significant reduction in the mean zones of inhibition following chemical and physical treatment of the supernatants (p = 0.0025). Acid, bacteriocins and H2O2 demonstrated potential for antagonism of the bacterial pathogens. These substances may augment each other rather that each working independently on the pathogens.

Knowledge of the abundance of bacterial species in vaginal communities will help us to better understand their role in health and disease. However, progress in this field has been limited because quantifying bacteria in natural specimens is an arduous process. We developed quantitative real-time PCR (qPCR) assays to facilitate assessments of bacterial abundance in vaginal specimens and evaluated the utility of these assays by measuring species abundance in patients whose vaginal floras were clinically described as normal, intermediate, or bacterial vaginosis (BV) as defined by Nugent's criteria. The qPCR measurements showed that Lactobacillus species were predominant in normal vaginal specimens and that high Lactobacillus crispatus and Lactobacillus jensenii abundance was specific to normal specimens, while Lactobacillus iners abundance was high in all categories including BV. The abundances of all non- Lactobacillus species were higher in BV specimens than in normal specimens. Prevotella species were prevalent in all specimens and represented a high percentage of total species in BV specimens. qPCR assays can be a useful tool for describing the structure of vaginal communities and elucidating their role in health and disease.

To determine whether different racial groups shared common types of vaginal microbiota, we characterized the composition and structure of vaginal bacterial communities in asymptomatic and apparently healthy Japanese women in Tokyo, Japan, and compared them with those of White and Black women from North America. The composition of vaginal communities was compared based on community profiles of terminal restriction fragments of 16S rRNA genes and phylogenetic analysis of cloned 16S rRNA gene sequences of the numerically dominant bacterial populations. The types of vaginal communities found in Japanese women were similar to those of Black and White women. As with White and Black women, most vaginal communities were dominated by lactobacilli, and only four species of Lactobacillus (Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri) were commonly found. Communities dominated by multiple species of lactobacilli were common in Japanese and White women, but rare in Black women. The incidence, in Japanese women, of vaginal communities with several non-Lactobacillus species at moderately high frequencies was intermediate between Black women and White women. The limited number of community types found among women in different ethnic groups suggests that host genetic factors, including the innate and adaptive immune systems, may be more important in determining the species composition of vaginal bacterial communities than are cultural and behavioral differences.

A 30-year-old Salvin's Amazon parrot (Amazona autumnalis salvini) with a history of a lifelong poor diet and inappropriate housing was presented in lateral recumbency to a veterinary teaching hospital for further evaluation. Radiological and ultrasonographic examination revealed a mild proventricular dilatation, mild hepatomegaly, signs of enteritis and airsacculitis. The main laboratory findings included a mild macrocytic hyperchromic anaemia, hypoglobulinaemia, decreased bile acids and increased alkaline phosphatase. In this bird a liver pathology was suspected because of the clinical, laboratory and ultrasonographic findings. The bird was treated with supportive care and metabolic aids. After initial improvement of the clinical signs, the bird's condition deteriorated and it died. Pathological findings revealed an endocarditis and myocarditis due to Lactobacillus jensenii and a bacteraemia. Endocarditis due to Lactobacillus sp. is a rare phenomenon in humans not yet described in animals. It is associated with severe underlying illnesses leading to translocation of otherwise non-pathogenic bacteria in the bloodstream. A similar pattern might be assumed in animals with compromised immunity.

Konjac glucomannan hydrolysate (GMH) was compared with inulin and glucose for its capacity to support the growth of probiotic bacteria but inhibit the growth of Candida albicans in vitro . The growth of lactic acid bacteria (LAB) was studied under aerobic, anaerobic and 5% CO2 conditions where the GMH progressively supported the growth of LAB as a function of concentration (0.1, 0.5, 1.0 and 2.0% w/v). In mixed cultures, GMH promoted the growth of LAB (even at concentrations as low as 0.1%) and consequently increased inhibition of C. albicans under anaerobic conditions, 5% CO2 or aerobic conditions. Inhibition of C. albicans growth was generally higher than that with glucose or inulin and, of the LAB strains, Lactobacillus jensenii exhibited most inhibition of the pathogen. The stimulatory effects of low concentrations of GMH on LAB and inhibition of C. albicans make these prebiotic and probiotic combinations a potential prophylactic or therapeutic agent for vaginal episodes. Key words: glucomannan hydrolysate (GMH), probiotic isolates, prebiotic, biocontrol, Candida albicans, bacterial vaginosis

Objective This study was undertaken to characterise the dominant species of Lactobacillus colonising the vagina of healthy pregnant women, to examine some of their phenotypic and genotypic properties, and to gain a better understanding of the potential role of species, which might be associated with infection-free status. Design A prospective descriptive cohort study. Setting Department of Obstetrics and Gynaecology, Medical University of Vienna and Medical School, Vienna, Austria. Sample A total of 200 women in the late first trimester of pregnancy without clinical signs of vaginal infection were included in the study. Of these, 126 women were found to have a normal vaginal flora based on Gram stain. Methods Culture probes from those 126 women were further processed for identification of Lactobacillus species. Overall, 168 colonies from 84 women were identified as belonging to the Lactobacillus genus. Based on the combined results of microbiological methods and genus-specific, multiplex, and species-specific polymerase chain reaction, lactobacilli were recovered from 72 women. Main outcome measures Identification of Lactobacillus species of the vaginal flora of healthy pregnant women. Results The most frequently occurring species were Lactobacillus crispatus and Lactobacillus gasseri, followed by Lactobacillus jensenii and Lactobacillus rhamnosus. Conclusions Our results may have implications on the composition and on the use of Lactobacillus preparations for the prevention of recurrent vaginal infection.

Background : Vaginal lactobacilli offer protection against recurrent urinary infections, bacterial vaginosis, and vaginal candidiasis. Objective : To characterise the isolated vaginal lactobacilli strains for their probiotic properties and to compare their probiotic potential. Methods : The Lactobacillus strains were isolated from vaginal samples by conventional culturing and identified by sequencing of the 16S rDNA fragment. Several functional properties were detected (production of hydrogen peroxide and lactic acid; antagonistic activity against Escherichia coli , Candida albicans , Candida glabrata , and Gardnerella vaginalis ; auto-aggregation and adhesiveness) as well as safety (haemolytic activity, antibiotic susceptibility, presence of transferrable resistance genes). Results : A total of 135 vaginal lactobacilli strains of three species, Lactobacillus crispatus (56%), Lactobacillus jensenii (26%), and Lactobacillus gasseri (18%) were characterised using several functional and safety tests. Most of L. crispatus (89%) and L. jensenii (86%) strains produced H 2 O 2 . The best lactic acid producers were L. gasseri (18.2±2.2 mg/ml) compared to L. crispatus (15.6±2.8 mg/ml) and L. jensenii (11.6±2.6 mg/ml) ( p< 0.0001; p< 0.0001, respectively). L. crispatus strains showed significantly higher anti- E. coli activity compared to L. jensenii . L. gasseri strains expressed significantly lower anticandidal activity compared to L. crispatus and L. jensenii ( p< 0.0001). There was no significant difference between the species in antagonistic activity against G. vaginalis . Nearly a third of the strains were able to auto-aggregate while all the tested strains showed a good ability to adhere to HeLa cells. None of the tested lactobacilli caused haemolysis. Although phenotypical resistance was not found to ampicillin, chloramphenicol, erythromycin, gentamycin, tetracycline, and vancomycin, the erm (B), tet (M), and tet (K) were detected in some strains. All strains were resistant to metronidazole, trimethoprim/sulfamethoxazole, and kanamycin. Conclusions : Our study revealed that the production of different antimicrobial metabolites is highly strain-specific and that the metabolites are not correlated with each other. L. crispatus displays better antagonistic activity against E. coli and Candida spp. than L. gasseri and L. jensenii ; therefore; a potential probiotic candidate could be found among L. crispatus strains.

Persistent infection with oncogenic Human Papillomavirus (HPV) is necessary for cervical carcinogenesis. Although evidence suggests that the vaginal microbiome plays a functional role in the persistence or regression of HPV infections, this has yet to be described in women with cervical intra-epithelial neoplasia (CIN). We hypothesised that increasing microbiome diversity is associated with increasing CIN severity. llumina MiSeq sequencing of 16S rRNA gene amplicons was used to characterise the vaginal microbiota of women with low-grade squamous intra-epithelial lesions (LSIL; n = 52), high-grade (HSIL; n = 92), invasive cervical cancer (ICC; n = 5) and healthy controls (n = 20). Hierarchical clustering analysis revealed an increased prevalence of microbiomes characterised by high-diversity and low levels of Lactobacillus spp. (community state type-CST IV) with increasing disease severity, irrespective of HPV status (Normal = 2/20,10%; LSIL = 11/52,21%; HSIL = 25/92,27%; ICC = 2/5,40%). Increasing disease severity was associated with decreasing relative abundance of Lactobacillus spp. The vaginal microbiome in HSIL was characterised by higher levels of Sneathia sanguinegens (P

The use of polymeric devices for controlled sustained delivery of drugs is a promising approach for the prevention of HIV-1 infection. Unfortunately, certain microbicides, when topically applied vaginally, may be cytotoxic to vaginal epithelial cells and the protective microflora present within the female genital tract. In this study, we evaluated the impact of hydroxychloroquine (HCQ)-loaded, reservoir-type, polyurethane intravaginal rings (IVRs) on the growth of Lactobacillus crispatus and Lactobacillus jensenii and on the viability of vaginal and ectocervical epithelial cells. The IVRs were fabricated using hot-melt injection molding and were capable of providing controlled release of HCQ for 24 days, with mean daily release rates of 17.01 ± 3.6 μg/ml in sodium acetate buffer (pH 4) and 29.45 ± 4.84 μg/ml in MRS broth (pH 6.2). Drug-free IVRs and the released HCQ had no significant effects on bacterial growth or the viability of vaginal or ectocervical epithelial cells. Furthermore, there was no significant impact on the integrity of vaginal epithelial cell monolayers, in comparison with controls, as measured by transepithelial electrical resistance. Overall, this is the first study to evaluate the effects of HCQ-loaded IVRs on the growth of vaginal flora and the integrity of vaginal epithelial cell monolayers.