RESUMEN
Background: Numerous bacteria are involved in the etiology of bacterial vaginosis (BV). Yet, current tests only focus on a select few. We therefore designed a new test targeting 22 BV-relevant species. Methods: Using 946 stored vaginal samples, a new qPCR test that quantitatively identifies 22 bacterial species was designed. The distribution and relative abundance of each species, α- and ß-diversities, correlation, and species co-existence were determined per sample. A diagnostic index was modeled from the data, trained, and tested to classify samples into BV-positive, BV-negative, or transitional BV. Results: The qPCR test identified all 22 targeted species with 95 - 100% sensitivity and specificity within 8 hours (from sample reception). Across most samples, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, Gardnerella vaginalis, Fannyhessea (Atopobium) vaginae, Prevotella bivia, and Megasphaera sp. type 1 were relatively abundant. BVAB-1 was more abundant and distributed than BVAB-2 and BVAB-3. No Mycoplasma genitalium was found. The inter-sample similarity was very low, and correlations existed between key species, which were used to model, train, and test a diagnostic index: MDL-BV index. The MDL-BV index, using both species and relative abundance markers, classified samples into three vaginal microbiome states. Testing this index on our samples, 491 were BV-positive, 318 were BV-negative, and 137 were transitional BV. Although important differences in BV status were observed between different age groups, races, and pregnancy status, they were statistically insignificant. Conclusion: Using a diverse and large number of vaginal samples from different races and age groups, including pregnant women, the new qRT-PCR test and MDL-BV index efficiently diagnosed BV within 8 hours (from sample reception), using 22 BV-associated species.
Asunto(s)
Gardnerella vaginalis , Lactobacillus , Microbiota , Reacción en Cadena en Tiempo Real de la Polimerasa , Vagina , Vaginosis Bacteriana , Femenino , Vaginosis Bacteriana/diagnóstico , Vaginosis Bacteriana/microbiología , Humanos , Vagina/microbiología , Microbiota/genética , Lactobacillus/aislamiento & purificación , Lactobacillus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto , Gardnerella vaginalis/aislamiento & purificación , Gardnerella vaginalis/genética , Adulto Joven , Sensibilidad y Especificidad , Prevotella/aislamiento & purificación , Prevotella/genética , Megasphaera/aislamiento & purificación , Megasphaera/genética , Actinobacteria/aislamiento & purificación , Actinobacteria/genética , Actinobacteria/clasificación , Persona de Mediana Edad , Lactobacillus crispatus/aislamiento & purificación , Lactobacillus crispatus/genética , Adolescente , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/clasificación , Embarazo , ARN Ribosómico 16S/genéticaRESUMEN
Female genital tract infection with high-risk human papilloma virus (HR-HPV) has the risk of developing into cervical cancer, and there is still a lack of effective therapeutic strategies. Probiotic intervention is considered as a potential intervention for HR-HPV, while exploration into living probiotic preparations for specific diseases remains limited and insufficient. This prospective controlled pilot study was conducted to observe the effect of intravaginal transplantation of a vaginal isolated natural probiotic strain, Lactobacillus crispatus chen-01, on the clearance of high-risk HPV infection. 100 women with high-risk HPV infection were enrolled and randomly divided into placebo group and probiotic treatment group, which received intravaginal transplantation of L. crispatus chen-01. Cervical exfoliated cells were collected 6 months later for detecting DNA load, typing of HPV, and cytological analysis. Our results showed that vaginal transplantation with L. crispatus chen-01 significantly reduced viral load of HPV, ameliorated HPV clearance rate, and improved vaginal inflammation state without causing obvious adverse reactions. Analysis of 16S rRNA sequencing revealed that L. crispatus chen-01 could effectively reconstitute the vaginal microbiota in women with high-risk HPV, which might be one of the underlying mechanisms of the beneficial effect of L. crispatus chen-01 transplantation. Our results suggested that vaginal transplantation of L. crispatus chen-01 might be a promising treatment for patients with high-risk HPV infection.
Asunto(s)
Lactobacillus crispatus , Infecciones por Papillomavirus , Probióticos , Vagina , Femenino , Humanos , Probióticos/uso terapéutico , Probióticos/administración & dosificación , Infecciones por Papillomavirus/terapia , Infecciones por Papillomavirus/virología , Proyectos Piloto , Lactobacillus crispatus/aislamiento & purificación , Vagina/microbiología , Vagina/virología , Adulto , Estudios Prospectivos , Administración Intravaginal , Carga Viral , Persona de Mediana Edad , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , MicrobiotaRESUMEN
Introduction: We assessed the in vitro anti-chlamydial activity of fresh vaginal secretions, deciphering the microbial and metabolic components able to counteract Chlamydia trachomatis viability. Methods: Forty vaginal samples were collected from a group of reproductive-aged women and their anti-chlamydial activity was evaluated by inhibition experiments. Each sample underwent 16S rRNA metabarcoding sequencing to determine the bacterial composition, as well as 1H-NMR spectroscopy to detect and quantify the presence of vaginal metabolites. Results: Samples characterized by a high anti-chlamydial activity were enriched in Lactobacillus, especially Lactobacillus crispatus and Lactobacillus iners, while not-active samples exhibited a significant reduction of lactobacilli, along with higher relative abundances of Streptococcus and Olegusella. Lactobacillus gasseri showed an opposite behavior compared to L. crispatus, being more prevalent in not-active vaginal samples. Higher concentrations of several amino acids (i.e., isoleucine, leucine, and aspartate; positively correlated to the abundance of L. crispatus and L. jensenii) lactate, and 4-aminobutyrate were the most significant metabolic fingerprints of highly active samples. Acetate and formate concentrations, on the other hand, were related to the abundances of a group of anaerobic opportunistic bacteria (including Prevotella, Dialister, Olegusella, Peptostreptococcus, Peptoniphilus, Finegoldia and Anaerococcus). Finally, glucose, correlated to Streptococcus, Lachnospira and Alloscardovia genera, emerged as a key molecule of the vaginal environment: indeed, the anti-chlamydial effect of vaginal fluids decreased as glucose concentrations increased. Discussion: These findings could pave the way for novel strategies in the prevention and treatment of chlamydial urogenital infections, such as lactobacilli probiotic formulations or lactobacilli-derived postbiotics.
Asunto(s)
Chlamydia trachomatis , Lactobacillus , ARN Ribosómico 16S , Vagina , Femenino , Humanos , Vagina/microbiología , ARN Ribosómico 16S/genética , Lactobacillus/aislamiento & purificación , Lactobacillus/genética , Lactobacillus/metabolismo , Chlamydia trachomatis/aislamiento & purificación , Adulto , Streptococcus/aislamiento & purificación , Adulto Joven , Lactobacillus crispatus/aislamiento & purificación , Infecciones por Chlamydia/microbiologíaRESUMEN
BACKGROUND: Bacterial vaginosis (BV) increases HIV acquisition risk, potentially by eliciting genital inflammation. After BV treatment, the vaginal administration of LACTIN-V, a live biotherapeutic containing the Lactobacillus crispatus strain CTV-05, reduced BV recurrence and vaginal inflammation; however, 3 months after product cessation, CTV-05 colonization was only sustained in 48% of participants. RESULTS: This nested sub-study in 32 participants receiving LACTIN-V finds that 72% (23/32) demonstrate clinically relevant colonization (CTV-05 absolute abundance > 106 CFU/mL) during at least one visit while 28% (9/32) of women demonstrate colonization resistance, even during product administration. Immediately prior to LACTIN-V administration, the colonization-resistant group exhibited elevated vaginal microbiota diversity. During LACTIN-V administration, colonization resistance was associated with elevated vaginal markers of epithelial disruption and reduced chemokines, possibly due to elevated absolute abundance of BV-associated species and reduced L. crispatus. Colonization permissive women were stratified into sustained and transient colonization groups (31% and 41% of participants, respectively) based on CTV-05 colonization after cessation of product administration. These groups also exhibited distinct genital immune profiles during LACTIN-V administration. CONCLUSIONS: The genital immune impact of LACTIN-V may be contingent on the CTV-05 colonization phenotype, which is in turn partially dependent on the success of BV clearance prior to LACTIN-V administration.
Asunto(s)
Lactobacillus crispatus , Vagina , Vaginosis Bacteriana , Humanos , Femenino , Vaginosis Bacteriana/microbiología , Vaginosis Bacteriana/inmunología , Vagina/microbiología , Adulto , Probióticos/administración & dosificación , Administración Intravaginal , Microbiota , Adulto Joven , FenotipoRESUMEN
Vaginal colonization by fungi may elicit genital inflammation and enhance the risk of adverse reproductive health outcomes, such as HIV acquisition. Cross-sectional studies have linked fungi with an absence of bacterial vaginosis (BV), but it is unclear whether shifts in vaginal bacteria alter the abundance of vaginal fungi. Vaginal swabs collected following topical metronidazole treatment for BV during the phase 2b, placebo-controlled trial of LACTIN-V, a Lactobacillus crispatus-based live biotherapeutic, were assayed with semi-quantitative PCR for the relative quantitation of fungi and key bacterial species and multiplex immunoassay for immune factors. Vaginal fungi increased immediately following metronidazole treatment for BV (adjusted P = 0.0006), with most of this increase attributable to Candida albicans. Vaginal fungi were independently linked to elevated levels of the proinflammatory cytokine interleukin (IL) 17A, although this association did not remain significant after correcting for multiple comparisons. Fungal relative abundance by semi-quantitative PCR returned to baseline levels within 1 month of metronidazole treatment and was not affected by LACTIN-V or placebo administration. Fungal abundance was positively associated with Lactobacillus species, negatively associated with BV-associated bacteria, and positively associated with a variety of proinflammatory cytokines and chemokines, including IL-17A, during and after study product administration. Antibiotic treatment for BV resulted in a transient expanded abundance of vaginal fungi in a subset of women which was unaffected by subsequent administration of LACTIN-V. Vaginal fungi were positively associated with Lactobacillus species and IL-17A and negatively associated with BV-associated bacteria; these associations were most pronounced in the longer-term outcomes.IMPORTANCEVaginal colonization by fungi can enhance the risk of adverse reproductive health outcomes and HIV acquisition, potentially by eliciting genital mucosal inflammation. We show that standard antibiotic treatment for bacterial vaginosis (BV) results in a transient increase in the absolute abundance of vaginal fungi, most of which was identified as Candida albicans. Vaginal fungi were positively associated with proinflammatory immune factors and negatively associated with BV-associated bacteria. These findings improve our understanding of how shifts in the bacterial composition of the vaginal microbiota may enhance proliferation by proinflammatory vaginal fungi, which may have important implications for risk of adverse reproductive health outcomes among women.
Asunto(s)
Metronidazol , Microbiota , Vagina , Vaginosis Bacteriana , Femenino , Humanos , Vagina/microbiología , Vagina/inmunología , Vaginosis Bacteriana/microbiología , Vaginosis Bacteriana/inmunología , Vaginosis Bacteriana/tratamiento farmacológico , Microbiota/efectos de los fármacos , Adulto , Candida albicans/inmunología , Candida albicans/efectos de los fármacos , Lactobacillus crispatus/aislamiento & purificación , Interleucina-17/metabolismo , Adulto Joven , Hongos/clasificación , Hongos/aislamiento & purificación , Hongos/efectos de los fármacos , Lactobacillus , Citocinas/metabolismo , Probióticos/administración & dosificación , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/efectos de los fármacosRESUMEN
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.
Asunto(s)
Candida albicans , Candidiasis Vulvovaginal , Extractos Vegetales , Simbióticos , Candida albicans/efectos de los fármacos , Extractos Vegetales/farmacología , Femenino , Humanos , Candidiasis Vulvovaginal/microbiología , Candidiasis Vulvovaginal/tratamiento farmacológico , Excreción Vaginal/microbiología , Biopelículas/efectos de los fármacos , Lactobacillus/efectos de los fármacos , Limosilactobacillus reuteri , Lactobacillus crispatus , Antifúngicos/farmacologíaRESUMEN
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.
Asunto(s)
Antibacterianos , Lactobacillus crispatus , Metronidazol , Probióticos , Vaginosis Bacteriana , Humanos , Femenino , Vaginosis Bacteriana/tratamiento farmacológico , Vaginosis Bacteriana/prevención & control , Vaginosis Bacteriana/microbiología , Metronidazol/administración & dosificación , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Adulto , Lactobacillus crispatus/fisiología , Probióticos/administración & dosificación , Resultado del Tratamiento , Recurrencia , Prevención Secundaria , Administración Intravaginal , Adulto Joven , Vagina/microbiología , Método Doble CiegoRESUMEN
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.
Asunto(s)
Cuello del Útero , Interacciones Microbiota-Huesped , Inmunidad Innata , Microbiota , Humanos , Femenino , Cuello del Útero/microbiología , Cuello del Útero/inmunología , Microbiota/inmunología , Interacciones Microbiota-Huesped/inmunología , Gardnerella vaginalis/inmunología , Lactobacillus crispatus/inmunología , Moco/inmunología , Moco/microbiología , Moco/metabolismo , Dispositivos Laboratorio en un ChipRESUMEN
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.
Asunto(s)
Lactobacillus , Microbiota , Vagina , Humanos , Femenino , Vagina/microbiología , Embarazo , Adulto , Estudios Longitudinales , Lactobacillus/aislamiento & purificación , Turquía/etnología , Trimestres del Embarazo , Adulto Joven , Etnicidad , Lactobacillus crispatus/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Lactobacillus crispatus , Vaginosis Bacteriana , Humanos , Femenino , Animales , Ratones , Gardnerella vaginalis/genética , Vaginosis Bacteriana/tratamiento farmacológico , Vaginosis Bacteriana/microbiología , Vagina/microbiología , Lactobacillus/metabolismoRESUMEN
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.
Asunto(s)
Candidiasis Vulvovaginal , Lactobacillus crispatus , Vaginosis Bacteriana , Humanos , Femenino , Vaginosis Bacteriana/diagnóstico , Candidiasis Vulvovaginal/diagnóstico , Vagina/microbiología , Gardnerella vaginalis/genética , Lactobacillus , Ácido LácticoRESUMEN
A microbiota vaginal é dominada pelo gênero Lactobacillus, mantendo um pH ácido crucial para a saúde. Alterações hormonais e a menopausa podem impactar essa microbiota. Estreptococos do Grupo B (EGB) são associados a infecções neonatais e podem colonizar a microbiota vaginal. A interação entre Lactobacillus e EGB é complexa, com resultados conflitantes em estudos in vitro e in vivo. O uso de probióticos contendo Lactobacillus pode ter benefícios, como alterar a positividade para EGB em um grupo que recebeu o tratamento. Contudo a replicabilidade deste resultado é limitada, e os mecanismos envolvidos nessa interação ainda são pouco elucidados. Portanto o objetivo desse estudo foi caracterizar cepas de EGB, avaliar sua interação com L. crispatus em diversas condições. Neste estudo foram selecionadas seis cepas, pertencentes aos sorotipos Ia, II, III e V. Essas cepas foram caracterizadas de acordo com o sorotipo capsular, presença de genes de virulência (hialuronidase, ß-citolisina/hemolisina, e ilhas de pili 1, 2a e 2b), resistência a antimicrobianos (penicilina, cefepima, vancomicina, eritromicina e clindamicina), curva de crescimento, Restriction Fragment Length Polymorphism (RFLP) e Multi-Locus Sequence Typing (MLST). O resultado de MLST identificou os sequence types ST1, ST23 ST28 e ST182, e os clonal complexes CC1, CC19 e CC23. Cepas possuem a maior parte dos genes de virulência testados. Somente uma cepa apresentou alguma resistência, no caso à eritromicina e clindamicina. Resultados que estão de acordo com a literatura como os principais descritos em isolados clínicos. Os ensaios de interação realizados foram: crescimento em co-cultura, formação de biofilme, ensaios de adesão e exclusão em cultura celular e por fim, análise do perfil proteico. Alguns efeitos de L. crispatus no EGB observados foram aumento na formação de biofilme de EGB e diminuição da sua proliferação quando em co-cultura. Contudo esses resultados variaram de acordo com a cepa de EGB. Nos ensaios de competição em cultura celular, a presença de L. crispatus levou à diminuição da adesão em uma cepa pertencente ao sorotipo III, e aumento de adesão em uma cepa pertencente ao sorotipo V. Quando a cultura de células recebeu tratamento prévio de L. crispatus, nos ensaios de exclusão, houve menor adesão de EGB. Contudo todos os efeitos observados são cepa-dependente; não foram encontradas associações entre o comportamento das cepas e seu sorotipo capsular ou fenótipo MLST. Estudos futuros podem elucidar as respostas ativadas na presença de microrganismos comensais
The vaginal microbiota is dominated by the genus Lactobacillus, which helps to maintain a crucial acidic pH for health. Hormonal changes and menopause can impact this microbiota. Group B Streptococcus (GBS) are associated with neonatal infections and can colonize the vaginal microbiota. The interaction between Lactobacillus and GBS is complex, with conflicting results in in vitro and in vivo studies. The use of probiotics containing Lactobacillus may have benefits, such as altering GBS positivity in a treated group. However, the replicability of this result is limited, and the mechanisms involved in this interaction are still poorly understood. The use of probiotics containing Lactobacillus may have some benefits, but further research is needed. These strains were characterized based on capsular serotype, presence of virulence genes (hyaluronidase, ß-cytolysin/hemolysin, and pili islands 1, 2a, and 2b), antimicrobial resistance (penicillin, cefepime, vancomycin, erythromycin, and clindamycin), growth curve, Restriction Fragment Length Polymorphism (RFLP), and Multi-Locus Sequence Typing (MLST) Therefore, the aim of this study was to characterize GBS strains, evaluate their interaction with L. crispatus under various conditions. Six strains were selected for this study, belonging to serotypes Ia, II, III, and V. MLST results identified sequence types ST1, ST23, ST28, and ST182, and clonal complexes CC1, CC19, and CC23. Most strains possessed the tested virulence genes, with only one strain showing resistance to erythromycin and clindamycin. These results are in line with the literature. Interaction assays included co-culture growth, biofilm formation, adhesion and exclusion assays in cell culture, and, finally, protein profile analysis. Some observed effects of L. crispatus on GBS included an increase in GBS biofilm formation and a decrease in GBS proliferation during co-culture. However, these results varied according to the GBS strain. GBS interaction with L. crispatus resulted in increased biofilm formation and decreased proliferation in co-culture. In cell culture competition assays, the presence of L. crispatus led to decreased adhesion in one GBS strain belonging to serotype III and increased adhesion in one strain belonging to serotype V. When cell culture received pre-treatment with L. crispatus, exclusion assays showed lower GBS adhesion. However, all observed effects are strain-dependent; no associations were found between strain behavior and capsular serotype or MLST phenotype. Future studies may elucidate the responses activated in the presence of commensal microorganisms
Asunto(s)
Streptococcus agalactiae/clasificación , Lactobacillus crispatus/clasificación , Técnicas In Vitro/métodos , Técnicas de Cultivo de Célula/métodos , Infecciones/complicacionesRESUMEN
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 2â336â109 bp, with a GC content of 37.04â% and 2303 ORFs, of which 1962 could be annotated. Analysis of the M247 mobilome, which accounts for 14â% of the whole genome, revealed the presence of: (i) Tn7088, a novel 14â105 bp long integrative and mobilizable element (IME) containing 16 ORFs; (ii) ΦM247, a novel 42â510 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.
Asunto(s)
Bacteriocinas , Lactobacillus crispatus , Bacteriocinas/genética , Bacteriófagos/genética , Lactobacillus crispatus/genética , Profagos/genéticaRESUMEN
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.
Asunto(s)
Candidiasis , Lactobacillus crispatus , Femenino , Humanos , Candida albicans , Células HeLa , Células Epiteliales/metabolismoRESUMEN
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).
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Lactobacillus crispatus , Probióticos , Humanos , Antibacterianos/uso terapéutico , Antibacterianos/farmacología , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/microbiología , Probióticos/uso terapéutico , Método Doble CiegoRESUMEN
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.
Asunto(s)
Infertilidad Femenina , Infertilidad Masculina , Lactobacillus crispatus , Torque teno virus , Masculino , Humanos , Femenino , Torque teno virus/genética , Semen , VaginaRESUMEN
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.
Asunto(s)
Lactobacillus crispatus , Probióticos , Vaginosis Bacteriana , Embarazo , Femenino , Humanos , Lactobacillus acidophilus , Lactobacillus/metabolismo , Gardnerella vaginalis , Mallas Quirúrgicas , Vaginosis Bacteriana/prevención & control , Vaginosis Bacteriana/microbiología , Ácido Láctico/metabolismo , Probióticos/farmacología , Parto ObstétricoRESUMEN
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.
Asunto(s)
Lactobacillus crispatus , Lactobacillus gasseri , Vaginosis Bacteriana , Femenino , Humanos , Adyuvantes Inmunológicos/uso terapéutico , Adyuvantes Farmacéuticos , Lactobacillus/fisiología , Metronidazol/uso terapéutico , Resultado del Tratamiento , Vagina/microbiología , Vaginosis Bacteriana/microbiologíaAsunto(s)
Lactobacillus crispatus , Probióticos , Femenino , Humanos , Vagina , Probióticos/uso terapéuticoRESUMEN
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.