Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human cervix chips.

Modulation of the cervix by steroid hormones and commensal microbiome play a central role in the health of the female reproductive tract. Here we describe organ-on-a-chip (Organ Chip) models that recreate the human cervical epithelial-stromal interface with a functional epithelial barrier and production of mucus with biochemical and hormone-responsive properties similar to living cervix. When Cervix Chips are populated with optimal healthy versus dysbiotic microbial communities (dominated by Lactobacillus crispatus and Gardnerella vaginalis, respectively), significant differences in tissue innate immune responses, barrier function, cell viability, proteome, and mucus composition are observed that are similar to those seen in vivo. Thus, human Cervix Organ Chips represent physiologically relevant in vitro models to study cervix physiology and host-microbiome interactions, and hence may be used as a preclinical testbed for development of therapeutic interventions to enhance women's health.

Response to Antibiotic Treatment of Bacterial Vaginosis Predicts the Effectiveness of LACTIN-V (Lactobacillus crispatus CTV-05) in the Prevention of Recurrent Disease.

OBJECTIVES: Live biotherapeutic products (LBPs) containing vaginal Lactobacillus crispatus are promising adjuvant treatments to prevent recurrent bacterial vaginosis (BV) but may depend on the success of initial antibiotic treatment. METHODS: A post hoc analysis of data collected during the phase 2b LACTIN-V randomized control trial (L. crispatus CTV-05) explored the impact of clinical BV cure defined as Amsel criteria 0 of 3 (excluding pH, per 2019 Food and Drug Administration guidance) 2 days after completion of treatment with vaginal metronidazole gel on the effectiveness of an 11-week LACTIN-V dosing regimen to prevent BV recurrence by 12 and 24 weeks. RESULTS: At enrollment, 88% of participants had achieved postantibiotic clinical BV cure. The effect of LACTIN-V on BV recurrence compared with placebo differed by initial clinical BV cure status. The LACTIN-V to placebo risk ratio of BV recurrence by 12 weeks was 0.56 (95% confidence interval, 0.35-0.77) among participants with initial clinical BV cure after metronidazole treatment and 1.34 (95% confidence interval, 0.47-2.23) among participants without postantibiotic clinical BV cure. Among women receiving LACTIN-V, those who had achieved postantibiotic clinical BV cure at enrollment reached higher levels of detectable L. crispatus CTV-05 compared with women failing to achieve postantibiotic clinical BV cure. CONCLUSIONS: LACTIN-V seems to only decrease BV recurrence in women with clinical cure of BV after initial antibiotic treatment. Future trials of LBPs should consider limiting enrollment to these women.

The vaginal microbiome composition during pregnancy in a region compromising different ethnic origins.

BACKGROUND: The vaginal microbiota plays a significant role in pregnancy outcomes and newborn health. Indeed, the composition and diversity of the vaginal microbiota can vary among different ethnic groups. Our study aimed to investigate the composition of the vaginal microbiome throughout the three trimesters of pregnancy and to identify any potential variations or patterns in the Turkish population compromising mixed ethnicities. METHOD: We conducted a longitudinal study to characterize the vaginal microbiota of pregnant women. The study included a total of 25 participants, and the samples were collected at each trimester: 11-13 weeks, 20-24 weeks and 28-34 weeks gestation. RESULTS: Lactobacillus species were consistently found to be dominant in the vaginal microbiota throughout all trimesters of pregnancy. Among Lactobacillus species, L. crispatus had the highest abundance in all trimesters (40.6%, 40.8% and 44.4%, respectively). L. iners was the second most prevalent species (28.5%, 31% and 25.04, respectively). Our findings reveal that the dominant composition of the vaginal microbiota aligns with the CST-type I, commonly observed in the European population. CONCLUSIONS: This suggests that there are shared mechanisms influencing the microbial communities in the vagina, which are likely influenced by factors such as genetics, lifestyle, and cultural behaviors rather than ethnicity alone. The complex interplay of these factors contributes to the establishment and maintenance of the vaginal microbiota during pregnancy. Understanding the underlying mechanisms and their impact on vaginal health across diverse populations is essential for improving pregnancy outcomes. The study was approved by the Koc University Ethical Committee (no:2019.093.IRB2.030) and registered at the clinical trials.

Synergistic Inhibition of Synbiotic Cultures among Lactobacilli and Plant Extracts against Vaginal Discharge Causing Candida albicans.

Vulvovaginal candidiasis (VVC) is the most common cause of vaginal discharge among women. The present study aimed to investigate the synergistic anticandidal effect of lactobacillus cultures supplemented with plant extracts. Among 600 isolates of lactic acid bacteria, 41 isolates exhibited inhibitory activity against Candida albicans ATCC10231. Six out of 41 cell-free supernatants demonstrated the most potent antibacterial and anticandidal activities. They also inhibited the clinical isolates of C. albicans, causing VVC and non-C. albicans. The synergistic effect between Lactobacillus crispatus 84/7 and Limosilactobacillus reuteri 89/4 was demonstrated by the lowest fractional inhibitory concentration index (FICI = 0.5). The synbiotic culture of bacterial combination, cultured with Jerusalem artichoke (H. tuberosus) extract, also exhibited the strongest inhibition against the tested C. albicans. Biofilm formation decreased after 12 h of incubation in the selected cell-free supernatants of this synbiotic culture. The anticandidal activity of crude extracts was lost after treatment with proteinase K and trypsin but not with heating conditions, suggesting that it may be a heat-stable substance. In conclusion, the combination of L. crispatus 84/7 and L. reuteri 89/4 with H. tuberosus may be a promising candidate for inhibiting Candida infection and biofilm formation, with the potential use as ingredients in vaginal biotherapeutic products.

Interpretation of vaginal metagenomic characteristics in different types of vaginitis.

Although vaginitis is closely related to vaginal microecology in females, the precise composition and functional potential of different types of vaginitis remain unclear. Here, metagenomic sequencing was applied to analyze the vaginal flora in patients with various forms of vaginitis, including cases with a clue cell proportion ranging from 1% to 20% (Clue1_20), bacterial vaginitis (BV), vulvovaginal candidiasis (VVC), and BV combined with VVC (VVC_BV). Our results identified Prevotella as an important biomarker between BV and Clue1_20. Moreover, a gradual decrease was observed in the relative abundance of shikimic acid metabolism associated with bacteria producing indole as well as a decline in the abundance of Gardnerella vaginalis in patients with BV, Clue1_20, and healthy women. Interestingly, the vaginal flora of patients in the VVC_BV group exhibited structural similarities to that of the VVC group, and its potentially functional characteristics resembled those of the BV and VVC groups. Finally, Lactobacillus crispatus was found in high abundance in healthy samples, greatly contributing to the stability of the vaginal environment. For the further study of L. crispatus, we isolated five strains of L. crispatus from healthy samples and evaluated their capacity to inhibit G. vaginalis biofilms and produce lactic acid in vitro to select the potential probiotic candidate for improving vaginitis in future clinical studies. Overall, we successfully identified bacterial biomarkers of different vaginitis and characterized the dynamic shifts in vaginal flora between patients with BV and healthy females. This research advances our understanding and holds great promise in enhancing clinical approaches for the treatment of vaginitis. IMPORTANCE: Vaginitis is one of the most common gynecological diseases, mostly caused by infections of pathogens such as Candida albicans and Gardnerella vaginalis. In recent years, it has been found that the stability of the vaginal flora plays an important role in vaginitis. Furthermore, the abundant Lactobacillus-producing rich lactic acid in the vagina provides a healthy acidic environment such as Lactobacillus crispatus. The metabolites of Lactobacillus can inhibit the colonization of pathogens. Here, we collected the vaginal samples of patients with bacterial vaginitis (BV), vulvovaginal candidiasis (VVC), and BV combined with VVC to discover the differences and relationships among the different kinds of vaginitis by metagenomic sequencing. Furthermore, because of the importance of L. crispatus in promoting vaginal health, we isolated multiple strains from vaginal samples of healthy females and chose the most promising strain with potential probiotic benefits to provide clinical implications for treatment strategies.

Lactobacillus crispatus CCFM1339 Inhibits Vaginal Epithelial Barrier Injury Induced by Gardnerella vaginalis in Mice.

The vaginal epithelial barrier, which integrates mechanical, immune, chemical, and microbial defenses, is pivotal in safeguarding against external pathogens and upholding the vaginal microecological equilibrium. Although the widely used metronidazole effectively curtails Gardnerella vaginalis, a key pathogen in bacterial vaginosis, it falls short in restoring the vaginal barrier or reducing recurrence rates. Our prior research highlighted Lactobacillus crispatus CCFM1339, a vaginally derived Lactobacillus strain, for its capacity to modulate the vaginal epithelial barrier. In cellular models, L. crispatus CCFM1339 fortified the integrity of the cellular monolayer, augmented cellular migration, and facilitated repair. Remarkably, in animal models, L. crispatus CCFM1339 substantially abated the secretion of the barrier disruption biomarker E-cadherin (from 101.45 to 82.90 pg/mL) and increased the anti-inflammatory cytokine IL-10 (35.18% vs. the model), consequently mitigating vaginal inflammation in mice. Immunological assays in vaginal tissues elucidated increased secretory IgA levels (from 405.56 to 740.62 ng/mL) and curtailed IL-17 gene expression. Moreover, L. crispatus CCFM1339 enhanced Lactobacilli abundance and attenuated Enterobacterium and Enterococcus within the vaginal microbiome, underscoring its potential in probiotic applications for vaginal barrier regulation.

The mobilome of Lactobacillus crispatus M247 includes two novel genetic elements: Tn7088 coding for a putative bacteriocin and the siphovirus prophage ΦM247.

Lactobacillus crispatus is a member of the vaginal and gastrointestinal human microbiota. Here we determined the complete genome sequence of the probiotic strain M247 combining Nanopore and Illumina technologies. The M247 genome is organized in one circular chromosome of 2 336 109 bp, with a GC content of 37.04 % and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14 % of the whole genome, revealed the presence of: (i) Tn7088, a novel 14 105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42 510 bp long siphovirus prophage containing 52 ORFs; (iii) three clustered regularly interspaced short palindromic repeats (CRISPRs); and (iv) 226 insertion sequences (ISs) belonging to 14 different families. Tn7088 has a modular organization including a mobilization module encoding FtsK homologous proteins and a relaxase, an integration/excision module coding for an integrase and an excisionase, and an adaptation module coding for a class I bacteriocin and homologous to the listeriolysin S (lls) locus of Listeria monocytogenes. Genome-wide homology search analysis showed the presence of Tn7088-like elements in 12 out of 23 L. crispatus complete public genomes. Mobilization and integration/excision modules are essentially conserved, while the adaptation module is variable since it is the target site for the integration of different ISs. Prophage ΦM247 contains genes for phage structural proteins, DNA replication and packaging, lysogenic and lytic cycles. ΦM247-like prophages are present in seven L. crispatus complete genomes, with sequence variability mainly due to the integration of ISs. PCR and sequencing showed that the Tn7088 IME excises from the M247 chromosome producing a circular form at a concentration of 4.32×10-5 copies per chromosome, and reconstitution of the Tn7088 chromosomal target site occurred at 6.65×10-4 copies per chromosome. The ΦM247 prophage produces an excised form and a reconstituted target site at a level of 3.90×10-5 and 2.48×10-5 copies per chromosome, respectively. This study identified two novel genetic elements in L. crispatus. Tn7088 represents the first example of an IME carrying a biosynthetic gene cluster for a class I bacteriocin in L. crispatus.

Protective Properties of S-layer Protein 2 from Lactobacillus crispatus 2029 against Candida albicans Infections.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni, Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human ß-defensin 3 in various epithelial cells. These findings support the anti-Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.

Study of the effect of Lactobacillus crispatus FSCDJY67L3 on Helicobacter Pylori eradication: a double-blind randomized controlled clinical trial.

Helicobacter pylori (H. pylori) is a gram-negative bacterium exhibiting high pathogenicity. Traditional antibiotic treatments are considered ineffective as the H. pylori resistance has increased. Recently, a quadruple therapy strategy of probiotics and antibiotics to eliminate H. pylori was proposed. Probiotics play a therapeutic role as supplements in this process. The present research screened a probiotic strain (Lactobacillus crispatus FSCDJY67L3) that co-aggregates strongly with H. pylori. L. crispatus FSCDJY67L3 was demonstrated to significantly reduce H. pylori load (14C breath test) in clinical trials with H. pylori-positive patients. The Gastrointestinal Symptom Rating Scale (GSRS) score decreased, indicating improvement in the gastrointestinal discomfort of patients. Furthermore, L. crispatus FSCDJY67L3 showed no change in the structure of the intestinal flora of patients. Routine blood indices and blood biochemical indices related to liver and kidney function were also not affected in the patients. Therefore, L. crispatus FSCDJY67L3 may be used clinically as a supplement for the treatment of H. pylori. Clinical Trial Registration: https://www.chictr.org.cn/, Chinese Clinical Trial Registry (ChiCTR2100053710).

Association between torquetenovirus in vaginal secretions and infertility: An exploratory metagenomic analysis.

PROBLEM: The association of viruses with infertility remains incompletely evaluated. METHOD OF STUDY: Vaginal secretions from 46 women seeking treatment in the Center for Reproductive Medicine and Infertility at Weill Cornell Medicine were tested for viruses by metagenomic analysis by lab personnel blinded to all clinical data. RESULTS: Torquetenovirus (TTV) was identified in 16 women, alphapapillomavirus in seven women and most were positive for bacteriophages. Twelve of the subjects were fertile and sought to freeze their oocytes for future implantation. These women were all negative for TTV. In contrast, 16 of the 34 women (47.1%) being treated for infertility were TTV-positive (p = .0035). Evaluating the women by cause of infertility, five of nine women (55.6%) whose male partner had inadequate sperm parameters and six of 14 women (42.9%) with defective ovulation were TTV positive (p = .0062 and p = .0171, respectively, vs. the fertile women). Alphapapillomavirus was identified in one (8.3%) fertile woman, five (35.7%) women with ovulation deficiency, and one (11.1%) woman with male factor infertility. These differences were not statistically significant. There were no differences in bacteriophage families or the presence of Lactobacillus phages between fertile or infertile women or between different causes of infertility. There was a negative association between TTV detection and Lactobacillus crispatus dominance in the vaginal microbiota (p = .0184), but no association between TTV detection and the presence of alphapapillomavirus or Candida species. CONCLUSION: Detection of TTV in the vagina might be a biomarker for specific causes of infertility.

Mesh and layered electrospun fiber architectures as vehicles for Lactobacillus acidophilus and Lactobacillus crispatus intended for vaginal delivery.

Bacterial vaginosis (BV) is a recurrent condition that affects millions of women worldwide. The use of probiotics is a promising alternative or an adjunct to traditional antibiotics for BV prevention and treatment. However, current administration regimens often require daily administration, thus contributing to low user adherence and recurrence. Here, electrospun fibers were designed to separately incorporate and sustain two lactic acid producing model organisms, Lactobacillus crispatus (L. crispatus) and Lactobacillus acidophilus (L. acidophilus). Fibers were made of polyethylene oxide and polylactic-co-glycolic acid in two different architectures, one with distinct layers and the other with co-spun components. Degradation of mesh and layered fibers was evaluated via mass loss and scanning electron microscopy. The results show that after 48 h and 6 days, cultures of mesh and layered fibers yielded as much as 108 and 109 CFU probiotic/mg fiber in total, respectively, with corresponding daily recovery on the order of 108 CFU/(mg·day). In addition, cultures of the fibers yielded lactic acid and caused a significant reduction in pH, indicating a high level of metabolic activity. The formulations did not affect vaginal keratinocyte viability or cell membrane integrity in vitro. Finally, mesh and layered probiotic fiber dosage forms demonstrated inhibition of Gardnerella, one of the most prevalent and abundant bacteria associated with BV, respectively resulting in 8- and 6.5-log decreases in Gardnerella viability in vitro after 24 h. This study provides initial proof of concept that mesh and layered electrospun fiber architectures developed as dissolving films may offer a viable alternative to daily probiotic administration.

Orally administrated Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 can restore the vaginal health of patients recovering from bacterial vaginosis.

Bacterial vaginosis (BV) is a common infection of the lower genital tract with a vaginal microbiome dysbiosis caused by decreasing of lactobacilli. Previous studies suggested that supplementation with live Lactobacillus may benefit the recovery of BV, however, the outcomes vary in people from different regions. Herein, we aim to evaluate the effectiveness of oral Chinese-origin Lactobacillus with adjuvant metronidazole (MET) on treating Chinese BV patients. In total, 67 Chinese women with BV were enrolled in this parallel controlled trial and randomly assigned to two study groups: a control group treated with MET vaginal suppositories for 7 days and a probiotic group treated with oral Lactobacillus gasseri TM13 and Lactobacillus crispatus LG55 as an adjuvant to MET for 30 days. By comparing the participants with Nugent Scores ≥ 7 and < 7 on days 14, 30, and 90, we found that oral administration of probiotics did not improve BV cure rates (72.73% and 84.00% at day 14, 57.14% and 60.00% at day 30, 32.14% and 48.39% at day 90 for probiotic and control group respectively). However, the probiotics were effective in restoring vaginal health after cure by showing higher proportion of participants with Nugent Scores < 4 in the probiotic group compared to the control group (87.50% and 71.43% on day 14, 93.75% and 88.89% on day 30, and 77.78% and 66.67% on day 90). The relative abundance of the probiotic strains was significantly increased in the intestinal microbiome of the probiotic group compared to the control group at day 14, but no significance was detected after 30 and 90 days. Also, the probiotics were not detected in vaginal microbiome, suggesting that L. gasseri TM13 and L. crispatus LG55 mainly acted through the intestine. A higher abundance of Prevotella timonensis at baseline was significantly associated with long-term cure failure of BV and greatly contributed to the enrichment of the lipid IVA synthesis pathway, which could aggravate inflammation response. To sum up, L. gasseri TM13 and L. crispatus LG55 can restore the vaginal health of patients recovering from BV, and individualized intervention mode should be developed to restore the vaginal health of patients recovering from BV. Clinical trial registration: https://classic.clinicaltrials.gov/ct2/show/, identifier NCT04771728.

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract.

The lower female reproductive tract is notoriously dominated by Lactobacillus species, among which Lactobacillus crispatus emerges for its protective and health-promoting activities. Although previous comparative genome analyses highlighted genetic and phenotypic diversity within the L. crispatus species, most studies have focused on the presence/absence of accessory genes. Here, we investigated the variation at the single nucleotide level within protein-encoding genes shared across a human-derived L. crispatus strain selection, which includes 200 currently available human-derived L. crispatus genomes as well as 41 chromosome sequences of such taxon that have been decoded in the framework of this study. Such data clearly pointed out the presence of intra-species micro-diversities that could have evolutionary significance contributing to phenotypical diversification by affecting protein domains. Specifically, two single nucleotide variations in the type II pullulanase gene sequence led to specific amino acid substitutions, possibly explaining the substantial differences in the growth performances and competition abilities observed in a multi-strain bioreactor culture simulating the vaginal environment. Accordingly, L. crispatus strains display different growth performances, suggesting that the colonisation and stable persistence in the female reproductive tract between the members of this taxon is highly variable.

Immunomodulatory effects of live and pasteurized Lactobacillus crispatus strain RIGLD-1 on Helicobacter pylori-triggered inflammation in gastric epithelial cells in vitro.

BACKGROUND: Helicobacter pylori infection is considered as the major risk factor for gastric adenocarcinoma. Today, the increasing emergence of antibiotic-resistant strains has drastically decreased the eradication rate of H. pylori infection. This study was aimed to investigate the inhibitory and modulatory effects of live and pasteurized Lactobacillus crispatus strain RIGLD-1 on H. pylori adhesion, invasion, and inflammatory response in AGS cell line. METHODS AND RESULTS: The probiotic potential and properties of L. crispatus were evaluated using several functional and safety tests. Cell viability of AGS cells exposed to varying concentrations of live and pasteurized L. crispatus was assessed by MTT assay. The adhesion and invasion abilities of H. pylori exposed to either live or pasteurized L. crispatus were examined by gentamycin protection assay. The mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR from coinfected AGS cells. ELISA was used for the detection of IL-8 secretion from treated cells. Both live and pasteurized L. crispatus significantly decreased H. pylori adhesion/invasion to AGS cells. In addition, both live and pasteurized L. crispatus modulated H. pylori-induced inflammation by downregulating the mRNA expression of IL-1ß, IL-6, IL-8, and TNF-α and upregulating the expression of IL-10, and TGF-ß cytokines in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with live and pasteurized L. crispatus. CONCLUSIONS: In conclusion, our findings demonstrated that live and pasteurized L. crispatus strain RIGLD-1 are safe, and could be suggested as a potential probiotic candidate against H. pylori colonization and inflammation.

Exploration of the protein-dependent mechanism of Lactobacillus crispatus GAI98322 to prevent recurrent cystitis.

OBJECTIVES: To elucidate the mechanism of Lactobacillus crispatus (L. crispatus) suppositories to prevent patients from recurrent cystitis (RC), independent from viable-Lactobacilli-bacteria- and acid-dependent ones such as hydrogen peroxide and lactate. METHODS: We used the GAI98322 strain of L. crispatus in all experiments and pH-matched. cell-free culture supernatant of L. crispatus (CFCS) was collected. The growth inhibitory activity and the biofilm formation inhibitory activity of the CFCS against uropathogenic Escherichia coli (UPEC), Extended Spectrum beta (ß) Lactamase producing (ESBL+) UPEC, and Pseudomonas aeruginosa (P. aeruginosa) was assessed by agar-disk diffusion tests and crystal violet assay. Also, CFCS was subjected to mass spectrometry to specify ingredients. RESULTS: The CFCS suppressed the proliferation of E. coli, ESBL + E. coli, and P. aeruginosa. Also, the CFCS at a concentration of 40% significantly impeded the biofilm formation of these three bacteria. The aggregation-promoting factor and Lysin was detected from CFCS. CONCLUSIONS: The cell-free supernatant from the GAI98322 strain of L. crispatus inhibits the growth/biofilm formation of broad pathogens by aggregation promoting factor and lysin, which may prevent hosts from RC regardless of the antimicrobial resistance of the pathogens and even under pH modulation.

Isolation and Characterization of Lactobacillus crispatus, Lactococcus lactis, and Carnobacterium divergens as Potential Probiotic Bacteria from Fermented Black and Green Olives (Olea europaea): An Exploratory Study.

Background: Table olives are becoming well recognized as a source of probiotic bacteria that might be used to create a health-promoting fermented food product by traditional procedures based on the activities of indigenous microbial consortia present in local environments. Methodology. In the present study, the characterization of probiotic bacteria isolated from mince, chunks, and brine of fermented green and black olives (Olea europaea) was done based on morphological, biochemical, and physiological characteristics. Results: Bacterial isolates demonstrated excellent survival abilities at 25, 37, and 45°C and at a variable range of pH. However, the optimum temperature is 37 and the optimum pH is 7 for all three isolates. An antimicrobial susceptibility pattern was found among these isolates through the disc diffusion method. Most of the isolates were susceptible to streptomycin, imipenem, and chloramphenicol, whereas, amoxicillin showed resistance to these isolates, and variable results were recorded for the rest of the antibiotics tested. The growth of the isolates was optimum with the supplementation of 3% NaCl and 0.3% bile salt. The isolated bacteria were able to ferment skimmed milk into yogurt, hence making it capable of producing organic acid. Conclusion: Isolates of Lactobacillus crispatus MB417, Lactococcus lactis MB418 from black olives, and Carnobacterium divergens MB421 from green olives were characterized as potential candidates for use as starter cultures to induce fermentation of other probiotic food products.

Lactobacillus crispatus-dominated vaginal microbiome and Acinetobacter-dominated seminal microbiome support beneficial ART outcome.

INTRODUCTION: Despite the considerable progress made in assisted reproductive technologies (ART), the implantation rate of transferred embryos remains low and in many cases, the reasons for failure remain unclear. We aimed to determine the potential impact of female and male partners' reproductive tract microbiome composition on ART outcome. MATERIAL AND METHODS: The ART couples (n = 97) and healthy couples (n = 12) were recruited into the study. The smaller healthy group underwent a careful selection according to their reproductive and general health criteria. Both vaginal and semen samples were subjected to 16S rDNA sequencing to reveal the bacterial diversity and identify distinct microbial community types. Ethics statement The study was approved by the Ethics Review Committee on Human Research of Tartu University, Tartu, Estonia (protocol no. 193/T-16) on 31 May 2010. Participation in the research was voluntary. Written informed consent was obtained from all study participants. RESULTS: The men with Acinetobacter-associated community who had children in the past, had the highest ART success rate (P < 0.05). The women with bacterial vaginosis vaginal microbiome community and with L. iners-predominant and L. gasseri-predominant microbiome had a lower ART success rate than women with the L. crispatus-predominant or the mixed lactic-acid-bacteria-predominant type (P < 0.05). The 15 couples where both partners had beneficial microbiome types had a superior ART success rate of 53%, when compared with the rest of the couples (25%; P = 0.023). CONCLUSIONS: Microbiome disturbances in the genital tract of both partners tend to be associated with couple's infertility as well as lower ART success levels and may thus need attention before the ART procedure. The incorporation of genitourinary microbial screening as a part of the diagnostic evaluation process may become routine for ART patients if our results are confirmed by other studies.

Milk and Dairy Consumption and Its Relationship With Abundance of Lactobacillus crispatus in the Vaginal Microbiota: Milk Intake and Vaginal Lactobacillus.

OBJECTIVES: Diet habits, such as low milk and dairy intake, have been associated with bacterial vaginosis. Thus, the authors compared vaginal Lactobacillus crispatus abundances in women with different molecularly defined community state types (CSTs) according to the consumption of milk and/or dairy products. METHODS: A total of 516 women from the 5 geographic regions of Brazil were included. Participants were interviewed with a structured questionnaire for assessment of milk and/or dairy intake. Vaginal samples were used for sequencing of V3-V4 regions of the 16S ribosomal RNA gene for further determination of L. crispatus relative abundance (RA) and clustering into 1 of the 5 CSTs (CSTI-CSTV), as firstly described by Ravel et al. (2011). The nonparametric Mann-Whitney test was used to compare L. crispatus RA within the most representative CSTs ( L. crispatus -dominant CSTI, Lactobacillus iners -dominant CSTIII, and Lactobacillus -depleted CSTIV) in this population, according to the frequency of milk and/or dairy intake. RESULTS: The prevalence of CSTI was 33.3% ( n = 172), CSTIII was 39% ( n = 201), and CSTIV was 27.7% ( n = 143). Among the participants with CSTIII, higher L. crispatus RA was observed for those who reported milk/dairy intake (median = 0.02; interquartile range = 0.01-0.09) than those with no consumption (median = 0.01; interquartile range = 0-0.03) ( p = .03). Such difference was not observed for participants with CSTI and CSTIV. CONCLUSIONS: Women with vaginal microbiota dominated by L. iners who consume milk and/or dairy present increased abundances of L. crispatus . Therefore, they could benefit from L. crispatus protective properties conferring greater temporal microbiota stability and, consequently, increased protection against infections.

Lactobacillus crispatus-loaded electrospun fibers yield viable and metabolically active bacteria that kill Gardnerella in vitro.

Bacterial vaginosis (BV) is a common condition that affects one-third of women worldwide. BV is characterized by low levels of healthy lactobacilli and an overgrowth of common anaerobes such as Gardnerella. Antibiotics for BV are administered orally or vaginally; however, approximately half of those treated will experience recurrence within 6 months. Lactobacillus crispatus present at high levels has been associated with positive health outcomes. To address the high recurrence rates following BV treatment, beneficial bacteria have been considered as an alternative or adjunct modality. This study aimed to establish proof-of-concept for a new long-acting delivery vehicle for L. crispatus. Here, it is shown that polyethylene oxide (PEO) fibers loaded with L. crispatus can be electrospun with poly(lactic-co-glycolic acid) (PLGA) fibers (ratio 1:1), and that this construct later releases L. crispatus as metabolically viable bacteria capable of lactic acid production and anti-Gardnerella activity. Probiotic-containing fibers were serially cultured in MRS (deMan, Rogosa, Sharpe) broth with daily media replacement and found to yield viable L. crispatus for at least 7 days. Lactic acid levels and corresponding pH values generally corresponded with levels of L. crispatus cultured from the fibers and strongly support the conclusion that fibers yield viable L. crispatus that is metabolically active. Cultures of L. crispatus-loaded fibers limited the growth of Gardnerella in a dilution-dependent manner during in vitro assays in the presence of cultured vaginal epithelial cells, demonstrating bactericidal potential. Exposure of VK2/E6E7 cells to L. crispatus-loaded fibers resulted in minimal loss of viability relative to untreated cells. Altogether, these data provide proof-of-concept for electrospun fibers as a candidate delivery vehicle for application of vaginal probiotics in a long-acting form.