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1.
Proc Natl Acad Sci U S A ; 119(33): e2117904119, 2022 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-35939684

RESUMO

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.


Assuntos
Infecções por Escherichia coli , Interferon Tipo I , Lactobacillus crispatus , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Catepsina D/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Interferon Tipo I/metabolismo , Camundongos , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/metabolismo
2.
Microbiome ; 10(1): 119, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35922830

RESUMO

BACKGROUND: The cervicovaginal (CV) microbiome is highly associated with vaginal health and disease in both pregnant and nonpregnant individuals. An overabundance of Gardnerella vaginalis (G. vaginalis) in the CV space is commonly associated with adverse reproductive outcomes including bacterial vaginosis (BV), sexually transmitted diseases, and preterm birth, while the presence of Lactobacillus spp. is often associated with reproductive health. While host-microbial interactions are hypothesized to contribute to CV health and disease, the mechanisms by which these interactions regulate CV epithelial function remain largely unknown. RESULTS: Using an in vitro co-culture model, we assessed the effects of Lactobacillus crispatus (L. crispatus) and G. vaginalis on the CV epithelial barrier, the immune mediators that could be contributing to decreased barrier integrity and the immune signaling pathways regulating the immune response. G. vaginalis, but not L. crispatus, significantly increased epithelial cell death and decreased epithelial barrier integrity in an epithelial cell-specific manner. A G. vaginalis-mediated epithelial immune response including NF-κB activation and proinflammatory cytokine release was initiated partially through TLR2-dependent signaling pathways. Additionally, investigation of the cytokine immune profile in human CV fluid showed distinctive clustering of cytokines by Gardnerella spp. abundance and birth outcome. CONCLUSIONS: The results of this study show microbe-specific effects on CV epithelial function. Altered epithelial barrier function through cell death and immune-mediated mechanisms by G. vaginalis, but not L. crispatus, indicates that host epithelial cells respond to bacteria-associated signals, resulting in altered epithelial function and ultimately CV disease. Additionally, distinct immune signatures associated with Gardnerella spp. or birth outcome provide further evidence that host-microbial interactions may contribute significantly to the biological mechanisms regulating reproductive outcomes. Video Abstract.


Assuntos
Lactobacillus crispatus , Nascimento Prematuro , Vaginose Bacteriana , Citocinas , Células Epiteliais , Feminino , Gardnerella vaginalis , Humanos , Imunidade , Recém-Nascido , Gravidez , Vagina/microbiologia , Vaginose Bacteriana/microbiologia
3.
Lancet Microbe ; 3(6): e435-e442, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35659905

RESUMO

BACKGROUND: Bacterial vaginosis might increase HIV risk by eliciting genital inflammation and epithelial barrier disruption, whereas vaginal Lactobacillus crispatus is associated with immune quiescence and HIV protection. We investigated the effect of a live biotherapeutic containing L crispatus CTV-05 (LACTIN-V) on genital immunology and key vaginal bacteria. METHODS: This substudy included women aged 18-45 years who participated in the randomised, placebo-controlled, phase 2b trial of LACTIN-V to reduce bacterial vaginosis recurrence, conducted at four universities and hospitals in the USA. Women with negative results for sexually transmitted infection, pregnancy, and urinary tract infection were provided a 5-day course of vaginal metronidazole 0·75% gel. Those who met at least three of four clinical Amsel criteria for bacterial vaginosis and had a Nugent score of 4-10 from Gram staining were eligible. Participants in the LACTIN-V trial were randomly assigned (2:1) to receive either LACTIN-V or placebo, applied vaginally once per day for 5 days during the first week and then twice per week for 10 more weeks. Follow-up visits occurred 4, 8, 12, and 24 weeks after enrolment. Soluble immune factors and the absolute abundance of bacterial taxa were assayed by mutliplex ELISA and quantitative PCR. The primary outcomes were vaginal levels of IL-1α and soluble E-cadherin at 24 weeks (ie, 13 weeks after treatment cessation). FINDINGS: Between Feb 21, 2020 and March 18, 2021, we characterised genital immune parameters and the vaginal microbiota in a subset of 66 highly adherent participants who were randomly selected, with no exclusion criteria, from those who had attended all study follow-up visits (n=166) in the larger LACTIN-V clinical trial (n=288). 32 (48%) participants received LACTIN-V and 34 (52%) received placebo. LACTIN-V treatment was significantly associated with lower concentrations of the proinflammatory cytokine IL-1α (ß coefficient 0·310, SE 0·149; p=0·042) and soluble E-cadherin (0·429, 0·199; p=0·035), a biomarker of epithelial barrier disruption. INTERPRETATION: Vaginal administration of LACTIN-V following standard bacterial vaginosis therapy resulted in a sustained reduction in genital inflammation and a biomarker of epithelial integrity. The potential of LACTIN-V to reduce HIV susceptibility merits further investigation. FUNDING: Canadian Institutes of Health Research and the National Institutes of Health National Institute of Allergy and Infectious Diseases.


Assuntos
Infecções por HIV , Lactobacillus crispatus , Vaginose Bacteriana , Bactérias , Caderinas/uso terapêutico , Canadá , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Metronidazol/uso terapêutico , Estados Unidos , Vagina/microbiologia , Vaginose Bacteriana/tratamento farmacológico
4.
Front Immunol ; 13: 905876, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35734171

RESUMO

Endometrial injury is the main cause of intrauterine adhesions (IUA), and there is currently no effective prevention and treatment. Immune cells play an important role in damage repair by sensing the change in the microenvironment. Exogenous CXCL12 can promote tissue regeneration and repair by recruiting immune cells, but its effect and possible mechanism on endometrial regeneration and repair have not been reported. In the present study, we constructed an engineered a Lactobacillus crispatus strain by transforming a pMG36e plasmid carrying a CXCL12 gene into the bacterium, and developed two animal models, the intrauterine adhesion mice with or without diabetes to evaluate the positive effects of this strain on the prevention of IUA after accepting intrauterine surgery in normal and diabetic mice. The results showed that vaginal application of L. crispatus-pMG36e-mCXCL12 strains significantly diminished the levels of pro-inflammatory factors interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) in serum and uterine tissues of IUA mice, and resulted in the inhibition of the inflammatory (toll-like receptor 4/nuclear factor-κb, TLR4/NF-κB) and fibrotic (transforming growth factor-ß1/smads, TGF-ß1/Smads) signalling pathways in the uterine tissues. The high-throughput sequencing results further indicated that treatment with L. crispatus-pMG36e-mCXCL12 strains greatly increased the abundance of Lactobacillus spp. and reduced that of the pathogenic Klebsiella spp. in IUA mice. Furthermore, among intrauterine adhesion mice with diabetes, we obtained similar results to non-diabetic mice, that is, L.crispatus-pMG36e-mCXCL12 significantly improved fibrosis and inflammation in the uterine cavity of diabetic mice, and restored the vaginal microbiota balance in diabetic mice. Therefore, we speculated that vaginal administration of L. crispatus-pMG36e-mCXCL12 strains can effectively alleviate intrauterine adhesions by restoring the microbial balance and reducing inflammation and fibrosis caused by surgery.


Assuntos
Quimiocina CXCL12 , Diabetes Mellitus Experimental , Lactobacillus crispatus , Aderências Teciduais , Doenças Uterinas , Animais , Quimiocina CXCL12/administração & dosagem , Feminino , Humanos , Inflamação/patologia , Camundongos , NF-kappa B , Aderências Teciduais/prevenção & controle , Doenças Uterinas/metabolismo , Doenças Uterinas/prevenção & controle
5.
Appl Microbiol Biotechnol ; 106(11): 4053-4064, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35612627

RESUMO

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.


Assuntos
Lactobacillus crispatus , Feminino , Glicogênio/metabolismo , Glicosídeo Hidrolases , Humanos , Lactobacillus/metabolismo , Lactobacillus crispatus/genética , Lactobacillus crispatus/metabolismo , Vagina
6.
Sci Rep ; 12(1): 7926, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35562576

RESUMO

Preterm birth is a major cause of neonatal morbidity and mortality worldwide. Increasing evidence links the vaginal microbiome to the risk of spontaneous preterm labour that leads to preterm birth. The aim of this systematic review and network meta-analysis was to investigate the association between the vaginal microbiome, defined as community state types (CSTs, i.e. dominance of specific lactobacilli spp, or not (low-lactobacilli)), and the risk of preterm birth. Systematic review using PubMed, Web of Science, Embase and Cochrane library was performed. Longitudinal studies using culture-independent methods categorizing the vaginal microbiome in at least three different CSTs to assess the risk of preterm birth were included. A (network) meta-analysis was conducted, presenting pooled odds ratios (OR) and 95% confidence intervals (CI); and weighted proportions and 95% CI. All 17 studies were published between 2014 and 2021 and included 38-539 pregnancies and 8-107 preterm births. Women presenting with "low-lactobacilli" vaginal microbiome were at increased risk (OR 1.69, 95% CI 1.15-2.49) for delivering preterm compared to Lactobacillus crispatus dominant women. Our network meta-analysis supports the microbiome being predictive of preterm birth, where low abundance of lactobacilli is associated with the highest risk, and L. crispatus dominance the lowest.


Assuntos
Lactobacillus crispatus , Microbiota , Nascimento Prematuro , Feminino , Humanos , Recém-Nascido , Lactobacillus , Metanálise em Rede , Gravidez , Vagina
7.
Int J Mol Sci ; 23(10)2022 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-35628398

RESUMO

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.


Assuntos
Lactobacillus crispatus , Feminino , Glucose/metabolismo , Glicogênio/metabolismo , Humanos , Lactobacillus/metabolismo , Lactobacillus crispatus/genética , Lactobacillus crispatus/metabolismo , Vagina/metabolismo
8.
Microbiol Spectr ; 10(2): e0273321, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35266820

RESUMO

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.


Assuntos
Lactobacillus crispatus , Microbiota , Antibacterianos/farmacologia , Biodiversidade , Feminino , Humanos , Lactobacillus crispatus/genética , Vagina/microbiologia
9.
Benef Microbes ; 13(1): 83-94, 2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-35144524

RESUMO

Streptococcus pyogenes, a group A streptococcus, is the major bacterial pathogen responsible for acute bacterial infection of the human oropharynx and the causative agent of scarlet fever. Estimates of the global burden of S. pyogenes related diseases revealed 616 million cases of pharyngitis, and at least 517,000 deaths due to severe invasive diseases and sequelae. Here we describe Lactobacillus crispatus DSM25988 that was identified among hundreds of Lactobacillus strains (referring to all organisms that were classified as Lactobacillaceae until 2020) showing ability to prevent adhesion of S. pyogenes to Detroit 562 cells, and to exhibit a masking and co-aggregating effect on S. pyogenes in vitro. L. crispatus DSM25988 also inhibits invasion of cultured human epithelial pharyngeal cells by S. pyogenes. Competitive binding to fibronectin might be involved in the inhibition process. Antiviral activity of the L. crispatus DSM25988 cells were identified in an in vitro cell model demonstrating that L. crispatus effectively excludes viruses from epithelial cells using SARS-CoV2 proteins as a model. This finding points to the potential of DSM25988 to protect cells from virus infection. Biological activity is retained in heat treated cells. The heat-treated Lactobacillus strain was further developed into functional throat lozenges, wherein its biological activity is stably maintained in the formulation. Lozenges containing L. crispatus DSM25988 underwent testing in an uncontrolled, prospective user study in 44 subjects with symptoms of sore throat for a period of up to 14 days. The study data shows promising safety and efficacy of the medical device when used against symptoms of sore throat like scratchy feeling, hoarse voice and swallowing pain.


Assuntos
COVID-19 , Lactobacillus crispatus , Probióticos , Humanos , Lactobacillus crispatus/fisiologia , Estudos Prospectivos , RNA Viral , SARS-CoV-2 , Streptococcus pyogenes
10.
Bioengineered ; 13(2): 2981-2991, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35038957

RESUMO

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).


Assuntos
Gardnerella vaginalis/genética , Lactobacillus crispatus/genética , RNA Ribossômico 16S/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vaginose Bacteriana/diagnóstico , Adolescente , Adulto , China , Estudos de Coortes , Técnicas de Diagnóstico Obstétrico e Ginecológico , Feminino , Gardnerella vaginalis/isolamento & purificação , Humanos , Lactobacillus crispatus/isolamento & purificação , Pessoa de Meia-Idade , RNA Bacteriano/análise , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de RNA/métodos , Vaginose Bacteriana/microbiologia , Adulto Jovem
11.
Microbiome ; 10(1): 15, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35074009

RESUMO

BACKGROUND: Immunoglobulin A (IgA) plays an important role in maintaining a healthy intestinal microbiome, but little is known about the interaction between local immunoglobulins and the vaginal microbiome. We assessed immunoglobulins (unbound and bound to bacteria), their association with vaginal microbiota composition and the changes over time in 25 healthy women of reproductive age. RESULTS: In both Lactobacillus crispatus-dominated and non-L. crispatus-dominated microbiota, IgA and IgG (unbound and bound to bacteria) were higher during menses (T = 1) compared to day 7­11 (T = 2) and day 17­25 (T = 3) after menses onset. The majority of vaginal bacteria are coated with IgA and/or IgG. Women with L. crispatus-dominated microbiota have increased IgA coating of vaginal bacteria compared to women with other microbiota compositions, but contained less IgA per bacterium. Presence of a dominantly IgA-coated population at T = 2 and/or T = 3 was also strongly associated with L. crispatus-dominated microbiota. In women with non-L. crispatus-dominated microbiota, more bacteria were uncoated. Unbound IgA, unbound IgG, and bound IgG levels were not associated with microbiota composition. CONCLUSIONS: In conclusion, L. crispatus-dominated vaginal microbiota have higher levels of bacterial IgA coating compared to non-L. crispatus-dominated vaginal microbiota. Similar to its regulating function in the intestinal tract, we hypothesize that IgA is involved in maintaining L. crispatus-dominated microbiota in the female genital tract. This may play a role in L. crispatus-associated health benefits. Video abstract.


Assuntos
Lactobacillus crispatus , Microbiota , Bactérias , Feminino , Humanos , Imunoglobulina A , Vagina/microbiologia
12.
Artigo em Inglês | LILACS | ID: biblio-1369851

RESUMO

Introduction: The majority of pregnant women with a short cervix will deliver at term and, thus, may unnecessarily receive advanced monitoring and treatment. It is still necessary to define more accurately which sub-population of women with a short cervix is at elevated risk for early delivery. Objective: To determine if vaginal microbiome composition influenced the rate of spontaneous preterm birth in women with a short cervical length. Methods: In an exploratory, observational prospective study, vaginal secretions were obtained from 591 women at 21­24 week gestation. Vaginal microbiome composition was determined by analyzing the V1­V3 region of the bacterial 16S ribosomal RNA gene. Results: Lactobacillus crispatus was numerically dominant in the vagina in 41.7% of subjects, followed by L. iners in 32% and Gardnerella vaginalis in 12%. In women whose cervix was ≤25mm, the sensitivity to predict an spontaneous preterm birth was 11.8%. However, when L. crispatus was not the dominant vaginal bacterium, this sensitivity increased to 81.8%. Similarly, in women with a cervical length ≤30mm, the sensitivity to predict an spontaneous preterm birth increased from 21.7 to 78.3% when L. crispatus was not the dominant vaginal bacterium.In women with a prior spontaneous preterm birth and a cervix ≤25 or ≤30mm, L. crispatus dominance was also associated with a reduced rate of spontaneous preterm birth in the current pregnancy (p<0.001). Conclusion: In pregnant women with a cervix ≤25mm or ≤30mm, the risk for an spontaneous preterm birth is increased if L. crispatus is not dominant in the vagina.


Introdução: A maioria das mulheres grávidas com colo do útero curto dará à luz a termo e, portanto, pode receber desnecessariamente monitoramento e tratamento avançados. Permanece a necessidade de definir com mais precisão qual subpopulação de mulheres com colo do útero curto está em risco elevado de parto prematuro. Objetivo: Determinar se a composição do microbioma vaginal influenciou a taxa de parto prematuro espontâneo em mulheres com colo curto. Métodos: Em um estudo prospectivo exploratório observacional, os conteúdos vaginais foram obtidos de 591 mulheres com 21­24 semanas de gestação. A composição do microbioma vaginal foi determinada pela análise da região V1­V3 do gene de RNA ribossômico bacteriano 16S. Resultados: Lactobacilluscrispatus foi numericamente dominante na vagina em 41,7% dos indivíduos, seguido por L. iners em 32% e Gardnerella vaginalis em 12%. Em mulheres cujo colo do útero era <25 mm, a sensibilidade para prever uma taxa de parto prematuro espontâneo foi de 11,8%. No entanto, quando L. crispatus não era a bactéria vaginal dominante, essa sensibilidade aumentou para 81,8%. Da mesma forma, em mulheres com comprimento cervical <30 mm, a sensibilidade para prever uma taxa de parto prematuro espontâneo aumentou de 21,7 para 78,3% quando L. crispatus não era a bactéria vaginal dominante. Em mulheres com taxa de parto prematuro espontâneo anterior e colo do útero <25 ou <30 mm, a dominância de L. crispatus também foi associada a uma taxa reduzida de taxa de parto prematuro espontâneo na gravidez atual (p<0,001). Conclusão: Em mulheres grávidas com colo do útero <25 ou <30 mm, o risco de parto prematuro espontâneo é aumentado se L. crispatus não for dominante na vagina.


Assuntos
Humanos , Feminino , Gravidez , Vagina/microbiologia , Microbiota , Lactobacillus crispatus , Trabalho de Parto Prematuro , Estudos Prospectivos , Medida do Comprimento Cervical
13.
Appl Environ Microbiol ; 88(4): e0239921, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-34910558

RESUMO

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.


Assuntos
Hexosiltransferases , Lactobacillus crispatus , Microbiota , Feminino , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Lactobacillus crispatus/genética , Eletricidade Estática , Vagina
14.
Int J Mol Sci ; 22(24)2021 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-34948426

RESUMO

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.


Assuntos
Lactobacillus/genética , Proteínas Luminescentes/genética , Microrganismos Geneticamente Modificados , Probióticos , Vagina/microbiologia , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Lactobacillus crispatus/genética , Lactobacillus gasseri/genética , Lactobacillus plantarum
15.
Microbiol Spectr ; 9(3): e0107421, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34756073

RESUMO

The vaginal microbiome composition in humans is categorized based upon the degree to which one of four species of Lactobacillus is dominant (Lactobacillus crispatus, community state type I [CST I], Lactobacillus gasseri, CST II, Lactobacillus iners, CST III, and Lactobacillus jensenii, CST V). Women with a vaginal microbiome not dominated by one of the four Lactobacillus species tend to have a more diverse microbiome, CST IV. CSTs I, II, III, and V are common in North America and Europe and are associated with lower incidences of some pathogens, such as human immunodeficiency virus (HIV), human papillomavirus (HPV), and Gardnerella vaginalis. As a result, therapeutic interventions to change the composition of the vaginal microbiomes are under development. However, Homo sapiens is the only mammalian species which has high frequencies of Lactobacillus-dominated vaginal microbiomes. Here, we treated female nonhuman primates (NHPs) with regimens of metronidazole and high levels of L. crispatus to determine how well these animals could be colonized with L. crispatus, how this influenced the immunological milieu, and how Lactobacillus treatment influenced or was influenced by the endogenous vaginal microbiome. We find that NHPs can transiently be colonized with L. crispatus, that beta diversity and not the number of doses of L. crispatus or pretreatment with metronidazole predicts subsequent L. crispatus colonization, that L. crispatus does not alter the local immunological milieu, and that the vaginal microbiome composition was resilient, normalizing by 4 weeks after our manipulations. Overall, this study suggests these animals are not amenable to long-term L. crispatus colonization. IMPORTANCE NHPs have proven to be invaluable animal models for the study of many human infectious diseases. The use of NHPs to study the effect of the microbiome on disease transmission and susceptibility is limited due to differences between the native microbiomes of humans and NHPs. In particular, Lactobacillus dominance of the vaginal microbiome is unique to humans and remains an important risk factor in reproductive health. By assessing the extent to which NHPs can be colonized with exogenously applied L. crispatus to resemble a human vaginal microbiome and examining the effects on the vaginal microenvironment, we highlight the utility of NHPs in analysis of vaginal microbiome manipulations in the context of human disease.


Assuntos
Chlorocebus aethiops/microbiologia , Lactobacillus crispatus/crescimento & desenvolvimento , Macaca mulatta/microbiologia , Microbiota/genética , Vagina/microbiologia , Animais , Antibacterianos/farmacologia , Feminino , Humanos , Inflamação/patologia , Lactobacillus crispatus/metabolismo , Menstruação/fisiologia , Metronidazol/farmacologia
16.
Food Funct ; 12(24): 12535-12549, 2021 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-34812468

RESUMO

Long-term use of antibiotic growth promoter (AGP) in animal production is the main cause of antimicrobial resistance of pathogenic bacteria. Therefore, seeking alternatives to AGP is crucial for animal husbandry. Among all AGP alternatives, probiotics are promising candidates. In this study, two strains of lactic acid bacteria, L. johnsonii 3-1 and L. crispatus 7-4, were isolated from the feces of wild Gallus gallus, which exhibited obvious anti-pathogenic activity and improved the growth performance of broilers. Furthermore, we found that these two strains participated in the lipid metabolism of broilers by reducing the content of TC and TG in ileal epithelial cells and up-regulating the liver AMPKα/PPARα/CPT-1 pathway, which affects abdominal fat deposition. In summary, L. johnsonii 3-1 and L. crispatus 7-4 have the potential to be used as AGP substitutes and participate in the lipid metabolism of broilers to reduce abdominal fat deposition. Importantly, our study reveals for the first time that L. crispatus participates in liver lipid metabolism to reduce abdominal fat deposition in broilers.


Assuntos
Peso Corporal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Lactobacillus crispatus/metabolismo , Lactobacillus johnsonii/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Probióticos/farmacologia , Animais , Galinhas , Íleo/crescimento & desenvolvimento , Íleo/metabolismo , Modelos Animais , Probióticos/metabolismo
17.
Sci Rep ; 11(1): 23069, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-34845288

RESUMO

We compared the effect of commercial vaginal douching products on Lactobacillus crispatus, L. jensenii, L. gasseri, L. iners, E. coli, and immortalized vaginal epithelial cells (VK2). All studied douching products (vinegar, iodine and baking soda based) induced epithelial cell death, and all inhibited growth of E. coli. Co-culture of vaginal epithelial cells with any of the lactobacilli immediately following exposure to douching products resulted in a trend to less human cell death. However, co-culture of epithelial cells with L. iners was associated with higher production of IL6 and IL8, and lower IL1RA regardless of presence or type of douching solution. Co-culture with L. crispatus or L. jensenii decreased IL6 production in the absence of douches, but increased IL6 production after exposure to vinegar. Douching products may be associated with epithelial disruption and inflammation, and may reduce the anti-inflammatory effects of beneficial lactobacilli.


Assuntos
Epitélio/efeitos dos fármacos , Epitélio/microbiologia , Escherichia coli/efeitos dos fármacos , Lactobacillus/efeitos dos fármacos , Ducha Vaginal/efeitos adversos , Ácido Acético , Sobrevivência Celular , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Sistema Imunitário , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Iodo , Lactobacillus crispatus , Lactobacillus gasseri , Testes de Sensibilidade Microbiana , Risco , Bicarbonato de Sódio , Infecções Urinárias/etiologia , Infecções Urinárias/prevenção & controle , Vagina/efeitos dos fármacos
18.
J Microbiol ; 59(11): 1019-1030, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34724180

RESUMO

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.


Assuntos
Lactobacillus crispatus/fisiologia , Mycobacterium tuberculosis/fisiologia , Probióticos/administração & dosagem , Tuberculose/tratamento farmacológico , Vagina/microbiologia , Adolescente , Adulto , Animais , Antibiose , Feminino , Cobaias , Humanos , Intestinos/microbiologia , Lactobacillus crispatus/classificação , Lactobacillus crispatus/genética , Lactobacillus crispatus/isolamento & purificação , Masculino , Microbiota , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Filogenia , Probióticos/isolamento & purificação , Tuberculose/microbiologia , Adulto Jovem
19.
Sci Rep ; 11(1): 18152, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518588

RESUMO

Steroid hormones are one of the presumed modulators of Lactobacillus abundance in the vaginal epithelium. We set out to characterize the vaginal microbiome (VMB) and also provide an in-depth understanding of the relative contribution of estradiol (E2) and progesterone (P1) in shaping the vaginal microbiome of Nigerian women (n = 38) who experienced both uncomplicated term delivery and preterm delivery using samples longitudinally collected during pregnancy (17-21, 27-31, 36-41 weeks gestation) and 6 weeks postpartum. Vaginal swabs and blood samples were aseptically collected. Vaginal swabs were used for microbiome assessment using 16S ribosomal RNA (rRNA) gene sequencing. Blood samples were used for hormonal measurement using a competitive-based enzyme-linked immunosorbent assay (ELISA). Across several maternal covariates, maternal age, pregnancy status and delivery mode were not significantly associated with the vaginal microbiota whereas maternal E2 level (pE2 = 0.006, Omnibus), and P1 level (pP1 = 0.001, Omnibus) were significantly associated with the vaginal microbiome. E2 and P1 concentrations increased throughout pregnancy commensurately with increasing proportions of L. crispatus (pE2 = 0.036, pP1 = 0.034, Linear Mixed Model). An increasing trend of α-diversity was also observed as pregnancy progressed (pobserved ASV = 0.006, LMM). A compositional microbiome shift from Lactobacillus profile to non-Lactobacillus profile was observed in most postnatal women (pCST IV < 0.001, LMM). Analysis of our data shows a species-specific link between pregnancy steroid hormone concentration and L. crispatus abundance.


Assuntos
Hormônios/metabolismo , Lactobacillus crispatus/fisiologia , Adulto , Bactérias/isolamento & purificação , Biodiversidade , Parto Obstétrico , Feminino , Idade Gestacional , Humanos , Microbiota , Nigéria , Filogenia , Período Pós-Parto/fisiologia , Gravidez , Nascimento Prematuro/microbiologia , Especificidade da Espécie , Vagina/microbiologia , Adulto Jovem
20.
Int J Biol Macromol ; 189: 410-419, 2021 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34437917

RESUMO

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes.


Assuntos
Diferenciação Celular , Enterócitos/metabolismo , Lactobacillus crispatus/química , Glicoproteínas de Membrana/farmacologia , Vagina/microbiologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Impedância Elétrica , Enterócitos/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Lactase/genética , Lactase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sacarase/genética , Sacarase/metabolismo
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