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.

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.

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.

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.

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina.

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatuspulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

Characterization of a novel type of glycogen-degrading amylopullulanase from Lactobacillus crispatus.

Glycogen is one of the major carbohydrates utilized by the human vaginal microbiota, which is commonly dominated by Lactobacillus, especially L. crispatus. An in silico analysis predicted that a type I pullulanase was involved in glycogen degradation in L. crispatus. The biochemical and genetic properties of the pullulanase still need to be determined. Here, we de novo identified the glycogen (Glg)-utilization enzyme (named GlgU) from L. crispatus through a biochemical assay. GlgU was optimally active at acidic pH, approximately 4.0 ~ 4.5, and was able to hydrolyze glycogen into low-molecular-weight malto-oligosaccharides. Actually, GlgU was a type II pullulanase (amylopullulanase) with just one catalytic domain that possessed substrate specificity toward both α-1,4 and α-1,6-glucosidic bonds. Phylogenetically, GlgU was obviously divergent from the known amylases and pullulanases (including amylopullulanases) in lactobacilli. In addition, we confirmed the catalytic activity of glgU in a nonglycogen-utilizing lactobacilli strain, demonstrating the essential role of glgU in glycogen metabolism. Overall, this study characterized a novel type of amylopullulanases, contributing to the knowledge of the glycogen utilization mechanism of the dominant species of human vaginal microbiota. KEY POINTS: • GlgU was a type II pullulanase, not a type I pullulanase predicted before. • GlgU was able to completely hydrolyze glycogen into malto-oligosaccharides. • GlgU played a key role in the metabolism of extracellular glycogen.

Evaluation of Modulatory Activities of Lactobacillus crispatus Strains in the Context of the Vaginal Microbiota.

It has been widely reported that members of the genus Lactobacillus dominate the vaginal microbiota, which is represented by the most prevalent species Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners. L. crispatus is furthermore considered an important microbial biomarker due to its professed beneficial implications on vaginal health. In order to identify molecular mechanisms responsible for health-promoting activities that are believed to be elicited by L. crispatus, we performed in silico investigations of the intraspecies biodiversity of vaginal microbiomes followed by in vitro experiments involving various L. crispatus strains along with other vaginal Lactobacillus species mentioned above. Specifically, we assessed their antibacterial activities against a variety of pathogenic microorganisms that are associated with vaginal infections. Moreover, coculture experiments of L. crispatus strains showing the most antibacterial activity against different pathogens revealed distinct ecological fitness and competitive properties with regard to other microbial colonizers. Interestingly, we observed that even phylogenetically closely related L. crispatus strains possess unique features in terms of their antimicrobial activities and associated competitive abilities, which suggests that they exert marked competition and evolutionary pressure within their specific environmental niche. IMPORTANCE The human vaginal microbiota includes all microorganisms that colonize the vaginal tract. In this context, a vaginal microbiota dominated by Lactobacillus and specifically by Lactobacillus crispatus is considered a hallmark of health. The role of L. crispatus in maintaining host health is linked to its modulatory activity toward other members of the vaginal ecosystem and toward the host. In this study, in vitro experiments followed by genetic analyses of the mechanisms used by L. crispatus in colonizing the vaginal ecological niche, particularly in the production of different antimicrobial compounds, were evaluated, highlighting some intriguing novel aspects concerning the genetic variability of this species. Our results indicate that this species has adapted to its niche and may still undergo adaptation to enhance its competitiveness for niche colonization.

Log (Lactobacillus crispatus/ Gardnerella vaginalis): a new indicator of diagnosing bacterial vaginosis.

To explore a new marker which can detect bacterial vaginosis (BV) with high sensitivity and specificity quantitatively. According to the Nugent Score, vaginal secretions from study participants were divided into BV, healthy, and BV-intermediate groups. First, we compared the obvious differences and high abundance of bacteria in the three groups using 16S rRNA-sequencing, and screened out candidate markers. Then, quantitative detection of these candidate markers from the three groups was done using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), followed by evaluation of the sensitivity and specificity. Finally, we verified the new markers using clinical cases. Gardnerella vaginalis, Atopobium vaginae, Lactobacillus, Megasphaera were screened out by 16S rRNA-sequencing. RT-qPCR data were transformed and analyzed through ROC curves. PCR results for these bacteria were log-transformed using Lactobacillus crispatus as the numerator and other BV-related bacteria as the denominator. Four new indicators were found. Of these, log L. crispatus/G. vaginalis (L/G) <0 was the best indicator. The sensitivity, specificity, positive predictive value, and negative predictive value of our system were 93.5%, 97.2%, 96.6 and 94.6%, respectively. Combination of data for 16S rRNA-sequencing and RT-qPCR revealed four indicators for BV detection. Of these, log L/G < 0 was the best indicator. Creating a molecular-diagnostic system independent of the Nugent Score for BV could have an important impact on the clinical management of BV.Abbreviation: log L. crispatus/G. vaginalis (logL/G); Bacterial vaginosis (BV); vaginal secretions (VSs); polymerase chain reaction (PCR); rRNA-sequencing (rRNA-seq); real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR); operational taxonomic unit (OTU); non-metric multidimensional scaling (NMDS); receiver operating characteristic (ROC).

Characterization of a Novel Fructosyltransferase from Lactobacillus crispatus, InuCA, That Attaches to the Cell Surface by Electrostatic Interaction.

Fructosyltransferases (FTases), a group of carbohydrate-active enzymes, synthesize fructooligosaccharides (FOS) and fructans, which are promising prebiotics for human health. Here, we identified a novel FTase, InuCA, from Lactobacillus crispatus, a dominant species in the vaginal microbiota of human. InuCA was characterized by the shortest C terminus and the highest isoelectric point among the reported Lactobacillus FTases. InuCA was an inulosucrase and produced a series of FOS using sucrose as the substrate at a moderate temperature. Surprisingly, the C-terminal deletion mutant synthesized oligosaccharides with the fructosyl chain longer than that of the wild type, suggesting that the C-terminal part blocked the binding of long-chain receptor. Moreover, InuCA bound to the cell surface by electrostatic interaction, which was dependent on the environmental pH and represented a distinctive binding mode in FTases. The catalytic and structural properties of InuCA will contribute to FTase engineering and the knowledge of the adaptation of L. crispatus in the vaginal environment. IMPORTANCE L. crispatus is one of the most important species in human vaginal microbiotas, and its persistence is strongly negatively correlated with vaginal diseases. Our research reveals that a novel inulosucrase, InuCA, is present in L. crispatus. InuCA keeps the ability to synthesize prebiotic fructo-oligosaccharides, although it lacks a large part of the C-terminal region compared to other FTases. Remarkably, the short C terminus of InuCA blocks the transfructosylation activity for producing oligosaccharides with longer chains, which is meaningful for the directional modification of FTases and the oligosaccharide products. Besides the catalytic activity, InuCA is anchored on the cell surface, depending on the environmental pH, and also may be involved in the adhesion of L. crispatus to the vaginal epithelial cells. Since L. crispatus plays an essential role in the normal vaginal micro-ecosystem, the described work will be helpful to elucidate the functional genes and colonization mechanism of the dominant species.

Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers.

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.

Activity of Lactobacillus crispatus isolated from vaginal microbiota against Mycobacterium tuberculosis.

Tuberculosis, an infectious disease, is caused by Mycobacterium tuberculosis. It remains a significant public health issue around the globe, causing about 1.8 million deaths every year. Drug-resistant M. tuberculosis, including multi-drug-resistant (MDR), extremely-drug-resistant (XDR), and totally drug-resistant (TDR) M. tuberculosis, continues to be a threat to public health. In the case of antibiotic-resistant tuberculosis, the treatment effect of conventional antibiotics is low. Side effects caused by high doses over a long period are causing severe problems. To overcome these problems, there is an urgent need to develop a new anti-tuberculosis drug that is different from the existing compound-based antibiotics. Probiotics are defined as live microorganisms conferring health benefits. They can be potential therapeutic agents in this context as the effectiveness of probiotics against different infectious diseases has been well established. Here, we report that Lactobacillus crispatus PMC201 shows a promising effect on tuberculosis isolated from vaginal fluids of healthy Korean women. Lactobacillus crispatus PMC201 reduced M. tuberculosis H37Rv under co-culture conditions in broth and reduced M. tuberculosis H37Rv and XDR M. tuberculosis in macrophages. Lactobacillus crispatus PMC201 was not toxic to a guinea pig model and did not induce dysbiosis in a human intestinal microbial ecosystem simulator. Taken together, these results indicate that L. crispatus PMC201 can be a promising alternative drug candidate in the current tuberculosis drug regime. Further study is warranted to assess the in vivo efficacy and confirm the mode of action of L. crispatus PMC201.

Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens.

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.

Vaginal bacterial load in the second trimester is associated with early preterm birth recurrence: a nested case-control study.

OBJECTIVE: To assess the association between vaginal microbiome (VMB) composition and recurrent early spontaneous preterm birth (sPTB)/preterm prelabour rupture of membranes (PPROM). DESIGN: Nested case-control study. SETTING: UK tertiary referral hospital. SAMPLE: High-risk women with previous sPTB/PPROM <34+0 weeks' gestation who had a recurrence (n = 22) or delivered at ≥37+0 weeks without PPROM (n = 87). METHODS: Vaginal swabs collected between 15 and 22 weeks' gestation were analysed by 16S rRNA gene sequencing and 16S quantitative PCR. MAIN OUTCOME MEASURE: Recurrent early sPTB/PPROM. RESULTS: Of the 109 high-risk women, 28 had anaerobic vaginal dysbiosis, with the remainder dominated by lactobacilli (Lactobacillus iners 36/109, Lactobacillus crispatus 23/109, or other 22/109). VMB type and diversity were not associated with recurrence. Women with a recurrence, compared to those without, had a higher median vaginal bacterial load (8.64 versus 7.89 log10 cells/mcl, adjusted odds ratio [aOR] 1.90, 95% CI 1.01-3.56, P = 0.047) and estimated Lactobacillus concentration (8.59 versus 7.48 log10 cells/mcl, aOR 2.35, (95% CI 1.20-4.61, P = 0.013). A higher recurrence risk was associated with higher median bacterial loads for each VMB type after stratification, although statistical significance was reached only for L. iners domination (aOR 3.44, 95% CI 1.06-11.15, P = 0.040). Women with anaerobic dysbiosis or L. iners domination had a higher median vaginal bacterial load than women with a VMB dominated by L. crispatus or other lactobacilli (8.54, 7.96, 7.63, and 7.53 log10 cells/mcl, respectively). CONCLUSIONS: Vaginal bacterial load is associated with early sPTB/PPROM recurrence. Domination by lactobacilli other than L. iners may protect women from developing high bacterial loads. Future PTB studies should quantify vaginal bacteria and yeasts. TWEETABLE ABSTRACT: Increased vaginal bacterial load in the second trimester may be associated with recurrent early spontaneous preterm birth.

Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity.

The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.

A specific bacterial DNA signature in the vagina of Australian women in midpregnancy predicts high risk of spontaneous preterm birth (the Predict1000 study).

BACKGROUND: Intrauterine infection accounts for a quarter of the cases of spontaneous preterm birth; however, at present, it is not possible to efficiently identify pregnant women at risk to deliver preventative treatments. OBJECTIVE: This study aimed to establish a vaginal microbial DNA test for Australian women in midpregnancy that will identify those at increased risk of spontaneous preterm birth. STUDY DESIGN: A total of 1000 women with singleton pregnancies were recruited in Perth, Australia. Midvaginal swabs were collected between 12 and 23 weeks' gestation. DNA was extracted for the detection of 23 risk-related microbial DNA targets by quantitative polymerase chain reaction. Obstetrical history, pregnancy outcome, and demographics were recorded. RESULTS: After excluding 64 women owing to losses to follow-up and insufficient sample for microbial analyses, the final cohort consisted of 936 women of predominantly white race (74.3%). The overall preterm birth rate was 12.6% (118 births); the spontaneous preterm birth rate at <37 weeks' gestation was 6.2% (2.9% at ≤34 weeks' gestation), whereas the preterm premature rupture of the membranes rate was 4.2%. No single individual microbial target predicted increased spontaneous preterm birth risk. Conversely, women who subsequently delivered at term had higher amounts of Lactobacillus crispatus, Lactobacillus gasseri, or Lactobacillus jensenii DNA in their vaginal swabs (13.8% spontaneous preterm birth vs 31.2% term; P=.005). In the remaining women, a specific microbial DNA signature was identified that was strongly predictive of spontaneous preterm birth risk, consisting of DNA from Gardnerella vaginalis (clade 4), Lactobacillus iners, and Ureaplasma parvum (serovars 3 and 6). Risk prediction was improved if Fusobacterium nucleatum detection was included in the test algorithm. The final algorithm, which we called the Gardnerella Lactobacillus Ureaplasma (GLU) test, was able to detect women at risk of spontaneous preterm birth at <37 and ≤34 weeks' gestation, with sensitivities of 37.9% and 44.4%, respectively, and likelihood ratios (plus or minus) of 2.22 per 0.75 and 2.52 per 0.67, respectively. Preterm premature rupture of the membranes was more than twice as common in GLU-positive women. Adjusting for maternal demographics, ethnicity, and clinical history did not improve prediction. Only a history of spontaneous preterm birth was more effective at predicting spontaneous preterm birth than a GLU-positive result (odds ratio, 3.6). CONCLUSION: We have identified a vaginal bacterial DNA signature that identifies women with a singleton pregnancy who are at increased risk of spontaneous preterm birth and may benefit from targeted antimicrobial therapy.

Vaginal microbiome analysis of healthy women during different periods of gestation.

To assess the vaginal microbiome throughout full-term uncomplicated pregnancy, a longitudinal study was designed for 12 healthy women who had prepared to become pregnant and then delivered at term (38-42 weeks) without complications. The vaginal microbial community was studied at pre-pregnancy, 8-12, 24-28, 37-38 weeks of gestation, and puerperium, using hypervariable tag sequencing of the V3-V4 region of the 16S rRNA gene. Sequencing produced approximately 10 million reads on the Illumina MiSeq. Members of the Firmicutes phyla were prevailing before and during pregnancy periods, and the proportion was quite as Proteobacteria until puerperium. Lactobacillus genus was abundant before and during pregnancy, but post-delivery vaginal microflora variety turned diverse. The species-level analysis revealed that a healthy vaginal microbiome before or during pregnancy was prominently dominated by Lactobacillus crispatus. Furthermore, PCoA analysis revealed for differences in the bacterial community composition between the two levels of Lactobacillus species in pre-pregnancy and pregnancy period (PC1 contribution of 58.46%, PC3 contribution of 8.64%). Based on the taxonomic and PCoA analysis, we found that L. crispatus was dominant in the vaginal microflora of healthy women before or during pregnancy, but at the puerperium, the status changed leading to decreased abundance of protective Lactobacillus species that made vaginal micro-ecological barrier vulnerable to diseases. Additionally, vaginal pH was an important environmental property affecting the vaginal microbial community.

Characterization of the Extracellular Fructanase FruA in Lactobacillus crispatus and Its Contribution to Fructan Hydrolysis in Breadmaking.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) trigger symptoms of irritable bowel syndrome (IBS). Fructan degradation during bread making reduces FODMAPs in bread while maintaining the content of dietary fiber. This study explored the presence of the fructanases FruA in lactobacilli and characterized its use in bread making. FruA was exclusively present in vertebrate-adapted lactobacilli. In Lactobacillus crispatus DSM29598, FruA was located in cell wall fractions and includes a SLAP domain. FruA hydrolyzed levan or inulin; expression of fruA was not subject to catabolite repression. Fructans in bread were reduced by less than 50% in a straight dough process; conventional sourdough fermentation reduced fructans in bread by 65-70%. Sourdough fermentation with L. crispatus reduced fructans in bread by more than 90%. In conclusion, reduction of FODMAP by sourdough fermentation may improve tolerance in many IBS patients. Fermentation with FruA-expressing L. crispatus DSM29598 produces a low FODMAP bread.

Comparative Genomics of Lactobacillus crispatus from the Gut and Vagina Reveals Genetic Diversity and Lifestyle Adaptation.

Lactobacillus crispatus colonizes the human feces, human vagina, and the crops and ceca of chicken. To explore the genetic characteristics and evolutionary relationships of L. crispatus isolated from different niches, we selected 37 strains isolated from the human vagina (n = 17), human feces (n = 11), and chicken feces (n = 9), and used comparative genomics to explore the genetic information of L. crispatus from the feces and vagina. No significant difference was found in the three sources of genomic features such as genome size, GC content, and number of protein coding sequences (CDS). However, in a phylogenetic tree constructed based on core genes, vagina-derived L. crispatus and feces-derived strains were each clustered separately. Therefore, the niche exerted an important impact on the evolution of L. crispatus. According to gene annotation, the L. crispatus derived from the vagina possessed a high abundance of genes related to acid tolerance, redox reactions, pullulanase, and carbohydrate-binding modules (CBMs). These genes helped L. crispatus to better adapt to the acidic environment of the vagina and obtain more nutrients, maintaining its dominance in the vagina in competition with other strains. In feces-derived bacteria, more genes encoding CRISPR/Cas system, glycoside hydrolases (GHs) family, and tetracycline/lincomycin resistance genes were found to adapt to the complex intestinal environment. This study highlights the evolutionary relationship of L. crispatus strains isolated from the vagina and feces, and the adaptation of L. crispatus to the host environment.

Safety assessment of the candidate oral probiotic Lactobacillus crispatus YIT 12319: Analysis of antibiotic resistance and virulence-associated genes.

Lactobacillus crispatus YIT 12319 (LcY) was isolated from the oral cavity of a healthy subject as a new candidate probiotic with potential benefits for oral health. As a safety assessment of LcY, we performed an antibiotic susceptibility test and virulence-associated gene analysis using a draft genome sequence. Susceptibility to 15 antibiotics was analyzed according to the standard method of the International Dairy Federation/International Organization for Standardization, as recommended by the European Food Safety Authority. The results showed that the minimum inhibitory concentrations of LcY were not higher than those of other L. crispatus strains, which have not acquired resistance to any antibiotics, suggesting that LcY had no externally acquired transmissible antibiotic resistance genes. Analysis of virulence-associated genes using the draft genome of LcY found that there were fewer potential virulence-associated genes in LcY than in other probiotics. These findings suggest that LcY could be a candidate probiotic based on its safety profile.