Precision probiotics supplement strategy in aging population based on gut microbiome composition.

With the increasing prevalence of age-related chronic diseases burdening healthcare systems, there is a pressing need for innovative management strategies. Our study focuses on the gut microbiota, essential for metabolic, nutritional, and immune functions, which undergoes significant changes with aging. These changes can impair intestinal function, leading to altered microbial diversity and composition that potentially influence health outcomes and disease progression. Using advanced metagenomic sequencing, we explore the potential of personalized probiotic supplements in 297 older adults by analyzing their gut microbiota. We identified distinctive Lactobacillus and Bifidobacterium signatures in the gut microbiota of older adults, revealing probiotic patterns associated with various population characteristics, microbial compositions, cognitive functions, and neuroimaging results. These insights suggest that tailored probiotic supplements, designed to match individual probiotic profile, could offer an innovative method for addressing age-related diseases and functional declines. Our findings enhance the existing evidence base for probiotic use among older adults, highlighting the opportunity to create more targeted and effective probiotic strategies. However, additional research is required to validate our results and further assess the impact of precision probiotics on aging populations. Future studies should employ longitudinal designs and larger cohorts to conclusively demonstrate the benefits of tailored probiotic treatments.

Evidence of Lactobacillus strains shared between the female urinary and vaginal microbiota.

Lactobacillus species are common inhabitants of the 'healthy' female urinary and vaginal communities, often associated with a lack of symptoms in both anatomical sites. Given identification by prior studies of similar bacterial species in both communities, it has been hypothesized that the two microbiotas are in fact connected. Here, we carried out whole-genome sequencing of 49 Lactobacillus strains, including 16 paired urogenital samples from the same participant. These strains represent five different Lactobacillus species: L. crispatus, L. gasseri, L. iners, L. jensenii, and L. paragasseri. Average nucleotide identity (ANI), alignment, single-nucleotide polymorphism (SNP), and CRISPR comparisons between strains from the same participant were performed. We conducted simulations of genome assemblies and ANI comparisons and present a statistical method to distinguish between unrelated, related, and identical strains. We found that 50 % of the paired samples have identical strains, evidence that the urinary and vaginal communities are connected. Additionally, we found evidence of strains sharing a common ancestor. These results establish that microbial sharing between the urinary tract and vagina is not limited to uropathogens. Knowledge that these two anatomical sites can share lactobacilli in females can inform future clinical approaches.

Molecular characterization of vaginal microbiota using a new 22-species qRT-PCR test to achieve a relative-abundance and species-based diagnosis of bacterial vaginosis.

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.

Sensoproteomic Characterization of Lactobacillus Johnsonii-Fermented Pea Protein-Based Beverage: A Promising Strategy for Enhancing Umami and Kokumi Sensations while Mitigating Bitterness.

This study investigated the mechanism underlying the flavor improvement observed during fermentation of a pea protein-based beverage using Lactobacillus johnsonii NCC533. A combination of sensomics and sensoproteomics approach revealed that the fermentation process enriched or generated well-known basic taste ingredients, such as amino acids, nucleotides, organic acids, and dipeptides, besides six new taste-active peptide sequences that enhance kokumi and umami notes. The six new umami and kokumi enhancing peptides, with human recognition thresholds ranging from 0.046 to 0.555 mM, are produced through the degradation of Pisum sativum's storage protein. Our findings suggest that compounds derived from fermentation enhance umami and kokumi sensations and reduce bitterness, thus improving the overall flavor perception of pea proteins. In addition, the analysis of intraspecific variations in the proteolytic activity of L. johnsonii and the genome-peptidome correlation analysis performed in this study point at cell-wall-bound proteinases such as PrtP and PrtM as the key genes necessary to initiate the flavor improving proteolytic cascade. This study provides valuable insights into the molecular mechanisms underlying the flavor improvement of pea protein during fermentation and identifies potential future research directions. The results highlight the importance of combining fermentation and senso(proteo)mics techniques in developing tastier and more palatable plant-based protein products.

An insight into the vaginal microbiome of infertile women in Bangladesh using metagenomic approach.

Introduction: The dysbiosis of vaginal microbiota is recognized as a potential underlying factor contributing to infertility in women. This study aimed to compare the vaginal microbiomes of infertile and fertile women to investigate their relationship with infertility. Methods: Metagenomic analysis was conducted on samples from 5 infertile and 5 fertile individuals using both amplicon 16S and metagenomics shotgun sequencing methods. Results and discussion: In the infertile group, the bacterial community was primarily represented by three major bacterial genera: Lactobacillus (79.42%), Gardnerella (12.56%) and Prevotella (3.33%), whereas, the fertile group exhibited a more diverse composition with over 8 major bacterial genera, accompanied by significantly reduced abundance of Lactobacillus (48.79%) and Gardnerella (6.98%). At the species level, higher abundances of L. iners, L. gasseri and G. vaginalis were observed in the infertile group. Regarding the microbiome composition, only one fertile and two infertile subjects exhibited the healthiest Community State Types, CST-1, while CST-3 was observed among two infertile and one fertile subject, and CST-4 in three other fertile and one infertile subject. Overall, alpha diversity metrics indicated greater diversity and lower species richness in the control (fertile) group, while the infertile group displayed the opposite trend. However, beta-diversity analysis did not show distinct clustering of samples associated with any specific group; instead, it demonstrated CST-type specific clustering. Shotgun metagenomics further confirmed the dominance of Firmicutes, with a greater abundance of Lactobacillus species in the infertile group. Specifically, L. iners and G. vaginalis were identified as the most dominant and highly abundant in the infertile group. Fungi were only identified in the control group, dominated by Penicillium citrinum (62.5%). Metagenome-assembled genomes (MAGs) corroborated read-based taxonomic profiling, with the taxon L. johnsonii identified exclusively in disease samples. MAG identities shared by both groups include Shamonda orthobunyavirus, L. crispatus, Human endogenous retrovirus K113, L. iners, and G. vaginalis. Interestingly, the healthy microbiomes sequenced in this study contained two clusters, Penicillium and Staphylococcus haemolyticus, not found in the public dataset. In conclusion, this study suggests that lower species diversity with a higher abundance of L. iners, L. gasseri and G. vaginalis, may contribute to female infertility in our study datasets. However, larger sample sizes are necessary to further evaluate such association.

The vaginal microbial signatures of preterm birth woman.

To explore the differences of vaginal microbes in women with preterm birth (PTB), and to construct prediction model. We searched for articles related to vaginal microbiology in preterm women and obtained four 16S rRNA-sequence datasets. We analyzed that for species diversity and differences, and constructed a random forest model with 20 differential genera. We introduce an independent whole genome-sequencing (WGS) data for validation. In addition, we collected vaginal and cervical swabs from 33 pregnant women who delivered spontaneously full-term and preterm infants, performed WGS in our lab to further validate the model. Compared to term birth (TB) samples, PTB women vagina were characterized by a decrease in Firmicutes, Lactobacillus, and an increase in diversity accompanied by the colonization of pathogenic bacteria such as Gardnerella, Atopobium and Prevotella. Twenty genus markers, including Lactobacillus, Prevotella, Streptococcus, and Gardnerella performed well in predicting PTB, with study-to-study transfer validation and LODO validation, different gestation validation showing good results, and in two independent cohorts (external WGS cohorts and woman samples WGS cohorts) in which the accuracy was maintained. PTB women have unique vaginal microbiota characteristics. A predictive model of PTB was constructed and its value validated from multiple perspectives.

Anti-chlamydial activity of vaginal fluids: new evidence from an in vitro model.

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.

Comparative study of the gut microbial community structure of Spodoptera frugiperda and Spodoptera literal (Lepidoptera).

Background: Spodoptera frugiperda, the fall armyworm is a destructive invasive pest, and S. litura the tobacco cutworm, is a native species closely related to S. frugiperda. The gut microbiota plays a vital role in insect growth, development, metabolism and immune system. Research on the competition between invasive species and closely related native species has focused on differences in the adaptability of insects to the environment. Little is known about gut symbiotic microbe composition and its role in influencing competitive differences between these two insects. Methods: We used a culture-independent approach targeting the 16S rRNA gene of gut bacteria of 5th instar larvae of S. frugiperda and S. litura. Larvae were reared continuously on maize leaves for five generations. We analyzed the composition, abundance, diversity, and metabolic function of gut microbiomes of S. frugiperda and S. litura larvae. Results: Firmicutes, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in both species. Enterococcus, ZOR0006, Escherichia, Bacteroides, and Lactobacillus were the genera with the highest abundance in S. frugiperda. Enterococcus, Erysipelatoclostridium, ZOR0006, Enterobacter, and Bacteroides had the highest abundance in S. litura. According to α-diversity analysis, the gut bacterial diversity of S. frugiperda was significantly higher than that of S. litura. KEGG analysis showed 15 significant differences in metabolic pathways between S. frugiperda and S. litura gut bacteria, including transcription, cell growth and death, excretory system and circulatory system pathways. Conclusion: In the same habitat, the larvae of S. frugiperda and S. litura showed significant differences in gut bacterial diversity and community composition. Regarding the composition and function of gut bacteria, the invasive species S. frugiperda may have a competitive advantage over S. litura. This study provides a foundation for developing control strategies for S. frugiperda and S. litura.

Effects of pickle brine and glycine addition on biogenic amine production in pickle fermentation.

This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %-55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.

Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

Heterologous expression and antimicrobial potential of class II bacteriocins.

Gut bacteria are known to produce bacteriocins to inhibit the growth of other bacteria. Consequently, bacteriocins have attracted increased attention as potential microbiome-editing tools. In this study we examine the inhibitory spectrum of 75 class II bacteriocins against 48 representative gut microbiota species. The bacteriocins were heterologously expressed in Escherichia coli and evaluated in vitro, ex vivo and in vivo. In vitro assays revealed 22 bacteriocins to inhibit at least one species and showed selective inhibition patterns against species implicated in certain disorders and diseases. Three bacteriocins were selected for ex vivo assessment on mouse feces. Based on 16S rRNA sequencing of the cultivated feces we showed that the two bacteriocins: Actifencin (#13) and Bacteroidetocin A (#22) selectively inhibited the growth of Lactobacillus and Bacteroides, respectively. The probiotic: E. coli Nissle 1917 was engineered to express these two bacteriocins in mice. However, the selective inhibitory patterns found in the in vitro and ex vivo experiments could not be observed in vivo. Our study describes a methodology for heterologous high throughput bacteriocin expression and screening and elucidates the inhibitory patterns of class II bacteriocins on the gut microbiota.

Impact of culture medium on the interpretation of qRT-PCR data in HepG2 incubated with lactobacilli.

Recently, an increasing number of studies have investigated the mechanism of action of lactobacilli in the treatment of non-alcoholic fatty liver disease. Using four computational tools (NormFinder, geNorm, Delta Ct, and BestKeeper), six potential reference genes (RGs) were analyzed in the human liver cell line HepG2 cultivated 24 h in the presence of two strains of heat-killed lactobacilli, Limosilactobacillus reuteri E and Lactiplantibacillus plantarum KG4, respectively, in different cultivation media [Dulbecco´s Modified Eagle´s Medium (DMEM) high glucose or Roswell Park Memorial Institute (RPMI)]. The analysis revealed that the suitability of RG was similar between the two lactobacilli but quite different between the two media. The commonly used RGs, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase were the most unstable in DMEM high glucose. Normalization of the mRNA expression of the target gene encoding sterol regulatory element-binding protein 1c (SREBP-1c) to different RGs resulted in different expression profiles. This demonstrates that validation of candidate RGs under specific experimental conditions is crucial for the correct interpretation of quantitative polymerase chain reaction data. In addition, the choice of media has a profound impact on the effect of lactobacilli on lipogenesis at the gene expression level, as shown by the transcription factor SREBP-1c.

Behavioral transcriptomic effects of triploidy and probiotic therapy (Bifidobacterium, Lactobacillus, and Lactococcus mixture) on juvenile Chinook salmon (Oncorhynchus tshawytscha).

Aquaculturists use polyploid fish to maximize production albeit with some unintended consequences including compromised behaviors and physiological function. Given benefits of probiotic therapies (e.g., improved immune response, growth, and metabolism), we explored probiotic supplementation (mixture of Bifidobacterium, Lactobacillus, and Lactococcus), to overcome drawbacks. We first examined fish gut bacterial community composition using 16S metabarcoding (via principal coordinate analyses and PERMANOVA) and determined probiotics significantly impacted gut bacteria composition (p = 0.001). Secondly, we examined how a genomic disruptor (triploidy) and diet supplements (probiotics) impact gene transcription and behavioral profiles of hatchery-reared Chinook salmon (Oncorhynchus tshawytscha). Juveniles from four treatment groups (diploid-regular feed, diploid-probiotic feed, triploid-regular feed, and triploid-probiotic feed; n = 360) underwent behavioral assays to test activity, exploration, neophobia, predator evasion, aggression/sociality, behavioral sensitivity, and flexibility. In these fish, transcriptional profiles for genes associated with neural functions (neurogenesis/synaptic plasticity) and biomarkers for stress response and development (growth/appetite) were (i) examined across treatments and (ii) used to describe behavioral phenotypes via principal component analyses and general linear mixed models. Triploids exhibited a more active behavioral profile (p = 0.002), and those on a regular diet had greater Neuropeptide Y transcription (p = 0.02). A growth gene (early growth response protein 1, p = 0.02) and long-term neural development genes (neurogenic differentiation factor, p = 0.003 and synaptysomal-associated protein 25-a, p = 0.005) impacted activity and reactionary profiles, respectively. Overall, our probiotic treatment did not compensate for triploidy. Our research highlights novel applications of behavioral transcriptomics for identifying candidate genes and dynamic, mechanistic associations with complex behavioral repertoires.

Phosphate Uptake and Its Relation to Arsenic Toxicity in Lactobacilli.

The use of probiotic lactobacilli has been proposed as a strategy to mitigate damage associated with exposure to toxic metals. Their protective effect against cationic metal ions, such as those of mercury or lead, is believed to stem from their chelating and accumulating potential. However, their retention of anionic toxic metalloids, such as inorganic arsenic, is generally low. Through the construction of mutants in phosphate transporter genes (pst) in Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains, coupled with arsenate [As(V)] uptake and toxicity assays, we determined that the incorporation of As(V), which structurally resembles phosphate, is likely facilitated by phosphate transporters. Surprisingly, inactivation in Lc. paracasei of PhoP, the transcriptional regulator of the two-component system PhoPR, a signal transducer involved in phosphate sensing, led to an increased resistance to arsenite [As(III)]. In comparison to the wild type, the phoP strain exhibited no differences in the ability to retain As(III), and there were no observed changes in the oxidation of As(III) to the less toxic As(V). These results reinforce the idea that specific transport, and not unspecific cell retention, plays a role in As(V) biosorption by lactobacilli, while they reveal an unexpected phenotype for the lack of the pleiotropic regulator PhoP.

Mutualistic interactions of lactate-producing lactobacilli and lactate-utilizing Veillonella dispar: Lactate and glutamate cross-feeding for the enhanced growth and short-chain fatty acid production.

The human gut hosts numerous ecological niches for microbe-microbe and host-microbe interactions. Gut lactate homeostasis in humans is crucial and relies on various bacteria. Veillonella spp., gut lactate-utilizing bacteria, and lactate-producing bacteria were frequently co-isolated. A recent clinical trial has revealed that lactate-producing bacteria in humans cross-feed lactate to Veillonella spp.; however, their interspecies interaction mechanisms remain unclear. Veillonella dispar, an obligate anaerobe commonly found in the human gut and oral cavity, ferments lactate into acetate and propionate. In our study, we investigated the interaction between V. dispar ATCC 17748T and three representative phylogenetically distant strains of lactic acid bacteria, Lactobacillus acidophilus ATCC 4356T, Lacticaseibacillus paracasei subsp. paracasei ATCC 27216T, and Lactiplantibacillus plantarum ATCC 10241. Bacterial growth, viability, metabolism and gene level adaptations during bacterial interaction were examined. V. dispar exhibited the highest degree of mutualism with L. acidophilus. During co-culture of V. dispar with L. acidophilus, both bacteria exhibited enhanced growth and increased viability. V. dispar demonstrated an upregulation of amino acid biosynthesis pathways and the aspartate catabolic pathway. L. acidophilus also showed a considerable number of upregulated genes related to growth and lactate fermentation. Our results support that V. dispar is able to enhance the fermentative capability of L. acidophilus by presumably consuming the produced lactate, and that L. acidophilus cross-feed not only lactate, but also glutamate, to V. dispar during co-culture. The cross-fed glutamate enters the central carbon metabolism in V. dispar. These findings highlight an intricate metabolic relationship characterized by cross-feeding of lactate and glutamate in parallel with considerable gene regulation within both L. acidophilus (lactate-producing) and V. dispar (lactate-utilizing). The mechanisms of mutualistic interactions between a traditional probiotic bacterium and a potential next-generation probiotic bacterium were elucidated in the production of short-chain fatty acids.

Isolation, identification, and characterization of thermophilic lactic acid bacteria isolated from whey of Kars Kashar cheeses.

Kars Kashar cheese is an artisanal pasta-filata type cheese and geographically marked in Eastern Anatolia of Turkey. The aims of this research were to determine for the first time thermophilic lactic acid bacteria (LAB) of Kars Kashar cheese and characterize the technological properties of obtained isolates. In our research, a number of 15 samples of whey were collected from the different villages in Kars. These samples were incubated at 45 °C and used as the source material for isolating thermophilic LAB. A total of 250 colonies were isolated from thermophilic whey, and 217 of them were determined to be presumptive LAB based on their Gram staining and catalase test. A total of 170 isolates were characterized by their phenotypic properties and identified using the MALDI-TOF mass spectrometry method. Phenotypic identification of isolates displayed that Enterococcus and Lactobacillus were the predominant microbiota. According to MALDI-TOF MS identification, 89 isolates were identified as Enterococcus (52.35%), 57 isolates as Lactobacillus (33.53%), 23 isolates as Streptococcus (13.53%), and one isolate as Lactococcus (0.59%). All thermophilic LAB isolates were successfully identified to the species level and it has been observed that MALDI-TOF MS can be successfully used for the identification of selected LAB. The acidification and proteolytic activities of the isolated thermophilic LAB were examined, and the isolates designated for use as starter cultures were also genotypically defined.

Vaginal microbiota molecular profiling and diagnostic performance of artificial intelligence-assisted multiplex PCR testing in women with bacterial vaginosis: a single-center experience.

Background: Bacterial vaginosis (BV) is a most common microbiological syndrome. The use of molecular methods, such as multiplex real-time PCR (mPCR) and next-generation sequencing, has revolutionized our understanding of microbial communities. Here, we aimed to use a novel multiplex PCR test to evaluate the microbial composition and dominant lactobacilli in non-pregnant women with BV, and combined with machine learning algorithms to determine its diagnostic significance. Methods: Residual material of 288 samples of vaginal secretions derived from the vagina from healthy women and BV patients that were sent for routine diagnostics was collected and subjected to the mPCR test. Subsequently, Decision tree (DT), random forest (RF), and support vector machine (SVM) hybrid diagnostic models were constructed and validated in a cohort of 99 women that included 74 BV patients and 25 healthy controls, and a separate cohort of 189 women comprising 75 BV patients, 30 intermediate vaginal microbiota subjects and 84 healthy controls, respectively. Results: The rate or abundance of Lactobacillus crispatus and Lactobacillus jensenii were significantly reduced in BV-affected patients when compared with healthy women, while Lactobacillus iners, Gardnerella vaginalis, Atopobium vaginae, BVAB2, Megasphaera type 2, Prevotella bivia, and Mycoplasma hominis were significantly increased. Then the hybrid diagnostic models were constructed and validated by an independent cohort. The model constructed with support vector machine algorithm achieved excellent prediction performance (Area under curve: 0.969, sensitivity: 90.4%, specificity: 96.1%). Moreover, for subjects with a Nugent score of 4 to 6, the SVM-BV model might be more robust and sensitive than the Nugent scoring method. Conclusion: The application of this mPCR test can be effectively used in key vaginal microbiota evaluation in women with BV, intermediate vaginal microbiota, and healthy women. In addition, this test may be used as an alternative to the clinical examination and Nugent scoring method in diagnosing BV.

Tuning the Dynamic Reaction Balance of CRISPR/Cas12a and RPA in One Pot: A Key to Switch Nucleic Acid Quantification.

Excavating nucleic acid quantitative capabilities by combining clustered regularly interspaced short palindromic repeats (CRISPR) and isothermal amplification in one pot is of common interest. However, the mutual interference between CRISPR cleavage and isothermal amplification is the primary obstacle to quantitative detection. Though several works have demonstrated enhanced detection sensitivity by reducing the inhibition of CRISPR on amplification in one pot, few paid attention to the amplification process and even dynamic reaction processes between the two. Herein, we find that DNA quantification can be realized by regulating either recombinase polymerase amplification (RPA) efficiency or CRISPR/Cas12a cleaving efficiency (namely, tuning the dynamic reaction balance) in one pot. The sensitive quantification is realized by utilizing dual PAM-free crRNAs for CRISPR/Cas12a recognition. The varied RPA primer concentration with stabilized CRISPR systems significantly affects the amplification efficiency and quantitative performances. Alternatively, quantitative detection can also be achieved by stabilizing the amplification process while regulating the CRISPR/Cas12a concentration. The quantitative capability is proved by detecting DNA targets from Lactobacillus acetotolerans and SARS-CoV-2. The quantitative performance toward real samples is comparable to quantitative real-time PCR for detecting L. acetotolerans spiked in fermented food samples and SARS-CoV-2 clinical samples. We expect that the presented method will be a powerful tool for quantifying other nucleic acid targets.

Effect of stress urinary incontinence on vaginal microbial communities.

BACKGROUND: Postpartum women often experience stress urinary incontinence (SUI) and vaginal microbial dysbiosis, which seriously affect women's physical and mental health. Understanding the relationship between SUI and vaginal microbiota composition may help to prevent vaginal diseases, but research on the potential association between these conditions is limited. RESULTS: This study employed 16S rRNA gene sequencing to explore the association between SUI and vaginal dysbiosis. In terms of the vaginal microbiota, both species richness and evenness were significantly higher in the SUI group. Additionally, the results of NMDS and species composition indicated that there were differences in the composition of the vaginal microbiota between the two groups. Specifically, compared to postpartum women without SUI (Non-SUI), the relative abundance of bacteria associated with bacterial dysbiosis, such as Streptococcus, Prevotella, Dialister, and Veillonella, showed an increase, while the relative abundance of Lactobacillus decreased in SUI patients. Furthermore, the vaginal microbial co-occurrence network of SUI patients displayed higher connectivity, complexity, and clustering. CONCLUSION: The study highlights the role of Lactobacillus in maintaining vaginal microbial homeostasis. It found a correlation between SUI and vaginal microbiota, indicating an increased risk of vaginal dysbiosis. The findings could enhance our understanding of the relationship between SUI and vaginal dysbiosis in postpartum women, providing valuable insights for preventing bacterial vaginal diseases and improving women's health.

Cervical microbiota dysbiosis associated with high-risk Human Papillomavirus infection.

High-risk Human Papillomavirus (HR-HPV) genotypes, specifically HPV16 and HPV18, pose a significant risk for the development of cervical intraepithelial neoplasia and cervical cancer. In the multifaceted cervical microenvironment, consisting of immune cells and diverse microbiota, Lactobacillus emerges as a pivotal factor, wielding significant influence in both stabilizing and disrupting the microbiome of the reproductive tract. To analyze the distinction between the cervical microbiota and Lactobacillus-dominant/non-dominant status of HR-HPV and non-infected healthy women, sixty-nine cervical swab samples were analyzed, included 44 with HR-HPV infection and healthy controls. All samples were recruited from Human Papillomavirus-based cervical cancer screening program and subjected to 16s rRNA sequencing analysis. Alpha and beta diversity analyses reveal no significant differences in the cervical microbiota of HR-HPV-infected women, including 16 and 18 HPV genotypes, and those with squamous intraepithelial lesion (SIL), compared to a control group. In this study we identified significantly lower abundance of Lactobacillus mucosae in women with HR-HPV infection compared to the control group. Furthermore, changes in bacterial diversity were noted in Lactobacillus non-dominant (LND) samples compared to Lactobacillus-dominant (LD) in both HR-HPV-infected and control groups. LND samples in HR-HPV-infected women exhibited a cervical dysbiotic state, characterized by Lactobacillus deficiency. In turn, the LD HR-HPV group showed an overrepresentation of Lactobacillus helveticus. In summary, our study highlighted the distinctive roles of L. mucosae and L. helveticus in HR-HPV infections, signaling a need for further research to demonstrate potential clinical implications of cervical microbiota dysbiosis.