Species-level resolution for the vaginal microbiota with short amplicons.

Specific bacterial species have been found to play important roles in human vagina. Achieving high species-level resolution is vital for analyzing vaginal microbiota data. However, contradictory conclusions were yielded from different methodological studies. More comprehensive evaluation is needed for determining an optimal pipeline for vaginal microbiota. Based on the sequences of vaginal bacterial species downloaded from NCBI, we conducted simulated amplification with various primer sets targeting different 16S regions as well as taxonomic classification on the amplicons applying different combinations of algorithms (BLAST+, VSEARCH, and Sklearn) and reference databases (Greengenes2, SILVA, and RDP). Vaginal swabs were collected from participants with different vaginal microecology to construct 16S full-length sequenced mock communities. Both computational and experimental amplifications were performed on the mock samples. Classification accuracy of each pipeline was determined. Microbial profiles were compared between the full-length and partial 16S sequencing samples. The optimal pipeline was further validated in a multicenter cohort against the PCR results of common STI pathogens. Pipeline V1-V3_Sklearn_Combined had the highest accuracy for classifying the amplicons generated from both the NCBI downloaded data (84.20% ± 2.39%) and the full-length sequencing data (95.65% ± 3.04%). Vaginal samples amplified and sequenced targeting the V1-V3 region but merely employing the forward reads (223 bp) and classified using the optimal pipeline, resembled the mock communities the most. The pipeline demonstrated high F1-scores for detecting STI pathogens within the validation cohort. We have determined an optimal pipeline to achieve high species-level resolution for vaginal microbiota with short amplicons, which will facilitate future studies.IMPORTANCEFor vaginal microbiota studies, diverse 16S rRNA gene regions were applied for amplification and sequencing, which affect the comparability between different studies as well as the species-level resolution of taxonomic classification. We conducted comprehensive evaluation on the methods which influence the accuracy for the taxonomic classification and established an optimal pipeline to achieve high species-level resolution for vaginal microbiota with short amplicons, which will facilitate future studies.

Limosilactobacillus reuteri ameliorates preeclampsia in mice via improving gut dysbiosis and endothelial dysfunction.

BACKGROUND: As a leading cause of maternal and neonatal morbidity and mortality, preeclampsia (PE) remains enigmatic. We confirmed that PE is associated with gut dysbiosis. However, whether probiotics could improve PE via regulating gut microbiota remains unclear. Exploring specific probiotic that could alleviate PE by modulating gut microbiota and elucidating detailed mechanisms will be essential for treating PE. METHODS: PE model was induced by nitric oxide (NO) inhibition with L-NAME in mice, and treated with Limosilactobacillus reuteri (L. reuteri). 16 S rDNA analysis was conducted on feces from mice. In vitro and in vivo experiments were performed to explore the roles and mechanisms of L. reuteri in PE. RESULTS: NO inhibition by L-NAME induced PE and gut dysbiosis in mice, characterized by differential gut microbiome, reduced alpha diversity, and markedly decreased abundance of Lactobacillales and L. reuteri. Importantly, L. reuteri could improve PE induced by L-NAME in mice, and ameliorate the gut dysbiosis of PE mice, including restorative gut microbiota composition, increased alpha diversity, and upregulated L. reuteri content. Moreover, L. reuteri could improve NO synthesis, angiogenesis, inflammation and oxidative stress of PE mice. Consistently, L. reuteri could ameliorate NO synthesis, endothelial dysfunction and inflammation mediated by L-NAME in vitro. CONCLUSIONS: Our results reveal that L. reuteri could ameliorate PE induced by NO inhibition in mice via improving gut dysbiosis and endothelial dysfunction, supporting gut microbiota serving as therapeutic target for treating PE originated from endothelial dysfunction and L. reuteri protecting patients from PE via modulating gut microbiota and endothelial function.

Akkermansia muciniphila prevents cold-related atrial fibrillation in rats by modulation of TMAO induced cardiac pyroptosis.

BACKGROUND: Cold exposure is one of the most important risk factors for atrial fibrillation (AF), and closely related to the poor prognosis of AF patients. However, the mechanisms underlying cold-related AF are poorly understood. METHODS: Various techniques including 16S rRNA gene sequencing, fecal microbiota transplantation, and electrophysiological examination were used to determine whether gut microbiota dysbiosis promotes cold-related AF. Metabonomics were performed to investigate changes in fecal trimethylamine (TMA) and plasma trimethylamine N-oxide (TMAO) during cold exposure. The detailed mechanism underlying cold-related AF were examined in vitro. Transgenic mice were constructed to explore the role of pyroptosis in cold-related AF. The human cohort was used to evaluate the correlation between A. muciniphila and cold-related AF. FINDINGS: We found that cold exposure caused elevated susceptibility to AF and reduced abundance of Akkermansia muciniphila (A. muciniphila) in rats. Intriguingly, oral supplementation of A. muciniphila ameliorated the pro-AF property induced by cold exposure. Mechanistically, cold exposure disrupted the A. muciniphila, by which elevated the level of trimethylamine N-oxide (TMAO) through modulation of the microbial enzymes involved in trimethylamine (TMA) synthesis. Correspondingly, progressively increased plasma TMAO levels were validated in human subjects during cold weather. Raised TMAO enhanced the infiltration of M1 macrophages in atria and increased the expression of Casp1-p20 and cleaved-GSDMD, ultimately causing atrial structural remodeling. Furthermore, the mice with conditional deletion of caspase1 exhibited resistance to cold-related AF. More importantly, a cross-sectional clinical study revealed that the reduction of A. muciniphila abundance was an independent risk factor for cold-related AF in human subjects. INTERPRETATION: Our findings revealed a novel causal role of aberrant gut microbiota and metabolites in pathogenesis of cold-related AF, which raises the possibility of selectively targeting microbiota and microbial metabolites as a potential therapeutic strategy for cold-related AF. FUNDING: This work was supported by grants from the State Key Program of National Natural Science Foundation of China (No.81830012), and National Natural Science Foundation of China (No.82070336, No.81974024), Youth Program of the National Natural Science Foundation of China (No.81900374, No.81900302), and Excellent Young Medical Talents supporting project in the First Affiliated Hospital of Harbin Medical University (No. HYD2020YQ0001).

Probiotics are a good choice for the treatment of bacterial vaginosis: a meta-analysis of randomized controlled trial.

BACKGROUND: Bacterial vaginosis (BV) is one of the most common vaginal infectious diseases in female reproductive period. Although the existing view is that probiotic treatment may be one of the feasible methods for the treatment of BV, different intervention methods lead to different treatment results. Therefore, up-to-date and comprehensive evidence in this regard is essential for the development of intervention strategies. OBJECTIVE: This meta-analysis aims to systematically evaluate the role of probiotics in the treatment of BV in adult women. METHODS: We searched the databases of Embase, Cochrane Library, PubMed, Web of Science and ClinicalTrials.gov for Randomized Controlled Trials published until November 7, 2021. Meta-analysis was performed by Revman5.3 software to systematically evaluate the clinical efficacy of probiotics adjunctive therapy in the treatment of BV. The literatures were screened and evaluated according to the inclusion and exclusion criteria. Chi-square test was used to test the heterogeneity between trials. Random or Fixed effect models were used to analyze the cure rate of BV. RESULTS: Fourteen randomized controlled trials compared the efficacy of probiotics with antibiotic therapy (probiotics + antibiotics group) versus antibiotics alone or plus placebo (antibiotics (+ placebo) group) for BV [Risk Ratios (RR) = 1.23, 95% CI (1.05, 1.43), P = 0.009]. Three compared the efficacy of probiotics regimen (probiotics group) and antibiotics (antibiotics group) in the treatment of BV [RR = 1.12, 95% CI (0.60, 2.07), P = 0.72]. Another Three compared the efficacy of probiotics regimen (probiotics group) with placebo (placebo group) [RR = 15.20, 95% CI (3.87, 59.64), P < 0.0001]. CONCLUSION: Our meta-analysis suggests probiotics may play a positive role in the treatment of BV, but more strong evidence is needed.

Our meta-analysis found that probiotics may play an active role in adjuvant treatment of bacterial vaginosis by conventional antibiotic therapy. It was emphasized that oral administration of L. rhamnose was more effective than vaginal application of L. rhamnose in the treatment of bacterial vaginosis. The therapeutic effect of probiotics varies with the administration route and dosage of probiotics.

Congenitally underdeveloped intestine drives autism-related gut microbiota and behavior.

Autism spectrum disorder (ASD) is a neurological and developmental disorder accompanied by gut dysbiosis and gastrointestinal symptoms in most cases. However, the development of the autism-related gut microbiota and its relationship with intestinal dysfunction in ASD remain unclear. Using a valproic acid (VPA)-induced ASD mouse model, we showed a congenitally immature intestine of VPA-exposed mice accompanied by prominent oxidative stress and inflammation. Of note, the gut microbiota composition of VPA-exposed mice resembled that of control mice within 24 h after birth; however, their gut microbiota compositions differed on postnatal days 7 and 21. Oral administration of superoxide dismutase (SOD) to attenuate intestinal oxidative stress either before weaning or during juvenile restored the autism-associated gut microbiota, leading to the amelioration of autism-related behaviors. These findings collectively suggest the congenitally underdeveloped intestine as an early driving force shaping the autism-associated gut microbiota and host neurodevelopment through enhancing oxidative stress.