Antibiotic-free vaginal microbiota transplant with donor engraftment, dysbiosis resolution and live birth after recurrent pregnancy loss: a proof of concept case study.

Background: Vaginal dysbiosis covers imbalances in the vaginal microbiota, defined by altered composition of bacteria, viruses, and fungi and is associated with euploid pregnancy losses, premature birth, infertility, or bacterial vaginosis. A large proportion of women who have vaginal dysbiosis do not experience any symptoms. Antibiotics are the traditional treatment, recently combined with local probiotics in some cases. Vaginal Microbiota Transplantation (VMT) with eubiotic vaginal bacterial microbiota after antibiotic eradication of pathogens has successfully been performed in a case study with five patients, but no VMT has been performed without the use of antibiotics. Methods: This is a proof of concept case study. The patient was found to have vaginal dysbiosis at the RPL clinic at Copenhagen University Hospital Hvidovre, Denmark on the 23rd of June 2021. She was offered and accepted to receive experimental treatment in the form of a VMT as a compassionate use case. VMT is the transfer of cervicovaginal secretions (CVS) from a healthy donor with a Lactobacillus-dominant vaginal microbiome to a recipient with a dysbiotic vaginal microbiome. CVS is a mixture of e.g., mucus, bacteria, metabolites present in the vaginal canal. Potential donors were thoroughly screened for the absence of STIs, and the most suitable donor sample for the specific patient in this study was determined via an in vitro microbiome competition assay. Findings: A 30-year-old patient with one livebirth and a complicated pregnancy history of two stillbirths and 1 s trimester pregnancy loss in gestational weeks 27 (2019), 17 (2020) and 23 (2020) respectively with complaints of vaginal irritation and discharge that had aggravated in all her pregnancies. Her vaginal microbiome composition showed a 90% dominance of Gardnerella spp. After one VMT there was a complete shift in microbiome composition to 81.2% L. crispatus and 9% L. jensenii with a concurrent resolvement of vaginal symptoms. Single nucleotide polymorphism-analysis confirmed her microbiome to be of donor origin and it remain stable now 1.5 years after the VMT. Five months after the VMT she became pregnant and has successfully delivered a healthy baby at term. Interpretation: Here we report a successful VMT with confirmed donor strain engraftment followed by a successful pregnancy and delivery after a series of late pregnancy losses/stillbirths. Findings suggest that VMT is a potential treatment for severe vaginal dysbiosis. Further, larger studies are required. Funding: The study was partially funded (i.e., analysis costs) by Freya Biosciences Aps, Fruebjergvej, 2100 Copenhagen, Denmark.

Synthetic glycans control gut microbiome structure and mitigate colitis in mice.

Relative abundances of bacterial species in the gut microbiome have been linked to many diseases. Species of gut bacteria are ecologically differentiated by their abilities to metabolize different glycans, making glycan delivery a powerful way to alter the microbiome to promote health. Here, we study the properties and therapeutic potential of chemically diverse synthetic glycans (SGs). Fermentation of SGs by gut microbiome cultures results in compound-specific shifts in taxonomic and metabolite profiles not observed with reference glycans, including prebiotics. Model enteric pathogens grow poorly on most SGs, potentially increasing their safety for at-risk populations. SGs increase survival, reduce weight loss, and improve clinical scores in mouse models of colitis. Synthetic glycans are thus a promising modality to improve health through selective changes to the gut microbiome.

Bifidobacteriumbreve Bif195 Protects Against Small-Intestinal Damage Caused by Acetylsalicylic Acid in Healthy Volunteers.

BACKGROUND & AIMS: Enteropathy and small-intestinal ulcers are common adverse effects of nonsteroidal anti-inflammatory drugs such as acetylsalicylic acid (ASA). Safe, cytoprotective strategies are needed to reduce this risk. Specific bifidobacteria might have cytoprotective activities, but little is known about these effects in humans. We used serial video capsule endoscopy (VCE) to assess the efficacy of a specific Bifidobacterium strain in healthy volunteers exposed to ASA. METHODS: We performed a single-site, double-blind, parallel-group, proof-of-concept analysis of 75 heathy volunteers given ASA (300 mg) daily for 6 weeks, from July 31 through October 24, 2017. The participants were randomly assigned (1:1) to groups given oral capsules of Bifidobacterium breve (Bif195) (≥5 × 1010 colony-forming units) or placebo daily for 8 weeks. Small-intestinal damage was analyzed by serial VCE at 6 visits. The area under the curve (AUC) for intestinal damage (Lewis score) and the AUC value for ulcers were the primary and first-ranked secondary end points of the trial, respectively. RESULTS: Efficacy data were obtained from 35 participants given Bif195 and 31 given placebo. The AUC for Lewis score was significantly lower in the Bif195 group (3040 ± 1340 arbitrary units) than the placebo group (4351 ± 3195) (P = .0376). The AUC for ulcer number was significantly lower in the Bif195 group (50.4 ± 53.1 arbitrary units) than in the placebo group (75.2 ± 85.3 arbitrary units) (P = .0258). Twelve adverse events were reported from the Bif195 group and 20 from the placebo group. None of the events was determined to be related to Bif195 intake. CONCLUSIONS: In a randomized, double-blind trial of healthy volunteers, we found oral Bif195 to safely reduce the risk of small-intestinal enteropathy caused by ASA. ClinicalTrials.gov no: NCT03228589.

Lactobacillus paracasei CNCM I-3689 reduces vancomycin-resistant Enterococcus persistence and promotes Bacteroidetes resilience in the gut following antibiotic challenge.

Enterococci, in particular vancomycin-resistant enterococci (VRE), are a leading cause of hospital-acquired infections. Promoting intestinal resistance against enterococci could reduce the risk of VRE infections. We investigated the effects of two Lactobacillus strains to prevent intestinal VRE. We used an intestinal colonisation mouse model based on an antibiotic-induced microbiota dysbiosis to mimic enterococci overgrowth and VRE persistence. Each Lactobacillus spp. was administered daily to mice starting one week before antibiotic treatment until two weeks after antibiotic and VRE inoculation. Of the two strains, Lactobacillus paracasei CNCM I-3689 decreased significantly VRE numbers in the feces demonstrating an improvement of the reduction of VRE. Longitudinal microbiota analysis showed that supplementation with L. paracasei CNCM I-3689 was associated with a better recovery of members of the phylum Bacteroidetes. Bile salt analysis and expression analysis of selected host genes revealed increased level of lithocholate and of ileal expression of camp (human LL-37) upon L. paracasei CNCM I-3689 supplementation. Although a direct effect of L. paracasei CNCM I-3689 on the VRE reduction was not ruled out, our data provide clues to possible anti-VRE mechanisms supporting an indirect anti-VRE effect through the gut microbiota. This work sustains non-antibiotic strategies against opportunistic enterococci after antibiotic-induced dysbiosis.

Brain Structure and Response to Emotional Stimuli as Related to Gut Microbial Profiles in Healthy Women.

OBJECTIVE: Brain-gut-microbiota interactions may play an important role in human health and behavior. Although rodent models have demonstrated effects of the gut microbiota on emotional, nociceptive, and social behaviors, there is little translational human evidence to date. In this study, we identify brain and behavioral characteristics of healthy women clustered by gut microbiota profiles. METHODS: Forty women supplied fecal samples for 16S rRNA profiling. Microbial clusters were identified using Partitioning Around Medoids. Functional magnetic resonance imaging was acquired. Microbiota-based group differences were analyzed in response to affective images. Structural and diffusion tensor imaging provided gray matter metrics (volume, cortical thickness, mean curvature, surface area) as well as fiber density between regions. A sparse Partial Least Square-Discrimination Analysis was applied to discriminate microbiota clusters using white and gray matter metrics. RESULTS: Two bacterial genus-based clusters were identified, one with greater Bacteroides abundance (n = 33) and one with greater Prevotella abundance (n = 7). The Prevotella group showed less hippocampal activity viewing negative valences images. White and gray matter imaging discriminated the two clusters, with accuracy of 66.7% and 87.2%, respectively. The Prevotella cluster was associated with differences in emotional, attentional, and sensory processing regions. For gray matter, the Bacteroides cluster showed greater prominence in the cerebellum, frontal regions, and the hippocampus. CONCLUSIONS: These results support the concept of brain-gut-microbiota interactions in healthy humans. Further examination of the interaction between gut microbes, brain, and affect in humans is needed to inform preclinical reports that microbial modulation may affect mood and behavior.

Ecological robustness of the gut microbiota in response to ingestion of transient food-borne microbes.

Resident gut microbes co-exist with transient bacteria to form the gut microbiota. Despite increasing evidence suggesting a role for transient microbes on gut microbiota function, the interplay between resident and transient members of this microbial community is poorly defined. We aimed to determine the extent to which a host's autochthonous gut microbiota influences niche permissivity to transient bacteria using a fermented milk product (FMP) as a vehicle for five food-borne bacterial strains. Using conventional and gnotobiotic rats and gut microbiome analyses (16S rRNA genes pyrosequencing and reverse transcription qPCR), we demonstrated that the clearance kinetics of one FMP bacterium, Lactococcus lactis CNCM I-1631, were dependent on the structure of the resident gut microbiota. Susceptibility of the resident gut microbiota to modulation by FMP intervention correlated with increased persistence of L. lactis. We also observed gut microbiome configurations that were associated with altered stability upon exposure to transient bacteria. Our study supports the concept that allochthonous bacteria have transient and subject-specific effects on the gut microbiome that can be leveraged to re-engineer the gut microbiome and improve dysbiosis-related diseases.

Lactobacillus rhamnosus CNCMI-4317 Modulates Fiaf/Angptl4 in Intestinal Epithelial Cells and Circulating Level in Mice.

BACKGROUND AND OBJECTIVES: Identification of new targets for metabolic diseases treatment or prevention is required. In this context, FIAF/ANGPTL4 appears as a crucial regulator of energy homeostasis. Lactobacilli are often considered to display beneficial effect for their hosts, acting on different regulatory pathways. The aim of the present work was to study the effect of several lactobacilli strains on Fiaf gene expression in human intestinal epithelial cells (IECs) and on mice tissues to decipher the underlying mechanisms. SUBJECTS AND METHODS: Nineteen lactobacilli strains have been tested on HT-29 human intestinal epithelial cells for their ability to regulate Fiaf gene expression by RT-qPCR. In order to determine regulated pathways, we analysed the whole genome transcriptome of IECs. We then validated in vivo bacterial effects using C57BL/6 mono-colonized mice fed with normal chow. RESULTS: We identified one strain (Lactobacillus rhamnosus CNCMI-4317) that modulated Fiaf expression in IECs. This regulation relied potentially on bacterial surface-exposed molecules and seemed to be PPAR-γ independent but PPAR-α dependent. Transcriptome functional analysis revealed that multiple pathways including cellular function and maintenance, lymphoid tissue structure and development, as well as lipid metabolism were regulated by this strain. The regulation of immune system and lipid and carbohydrate metabolism was also confirmed by overrepresentation of Gene Ontology terms analysis. In vivo, circulating FIAF protein was increased by the strain but this phenomenon was not correlated with modulation Fiaf expression in tissues (except a trend in distal small intestine). CONCLUSION: We showed that Lactobacillus rhamnosus CNCMI-4317 induced Fiaf expression in human IECs, and increased circulating FIAF protein level in mice. Moreover, this effect was accompanied by transcriptome modulation of several pathways including immune response and metabolism in vitro.

Fate, activity, and impact of ingested bacteria within the human gut microbiota.

The human gut contains a highly diverse microbial community that is essentially an open ecosystem, despite being deeply embedded within the human body. Food-associated fermentative bacteria, including probiotics, are major sources of ingested bacteria that may temporarily complement resident microbial communities, thus forming part of our transient microbiome. Here, we review data on the fate and activity of ingested bacteria and, in particular, lactobacilli and bifidobacteria in the gastrointestinal (GI) tract and their impact on the composition and metabolism of the gut microbiome with a focus on data from clinical studies. In addition, we discuss the mechanisms involved and the potential impact on the host's health.

Modulation of gut microbiota during probiotic-mediated attenuation of metabolic syndrome in high fat diet-fed mice.

Structural disruption of gut microbiota and associated inflammation are considered important etiological factors in high fat diet (HFD)-induced metabolic syndrome (MS). Three candidate probiotic strains, Lactobacillus paracasei CNCM I-4270 (LC), L. rhamnosus I-3690 (LR) and Bifidobacterium animalis subsp. lactis I-2494 (BA), were individually administered to HFD-fed mice (10(8) cells day(-1)) for 12 weeks. Each strain attenuated weight gain and macrophage infiltration into epididymal adipose tissue and markedly improved glucose-insulin homeostasis and hepatic steatosis. Weighted UniFrac principal coordinate analysis based on 454 pyrosequencing of fecal bacterial 16S rRNA genes showed that the probiotic strains shifted the overall structure of the HFD-disrupted gut microbiota toward that of lean mice fed a normal (chow) diet. Redundancy analysis revealed that abundances of 83 operational taxonomic units (OTUs) were altered by probiotics. Forty-nine altered OTUs were significantly correlated with one or more host MS parameters and were designated 'functionally relevant phylotypes'. Thirteen of the 15 functionally relevant OTUs that were negatively correlated with MS phenotypes were promoted, and 26 of the 34 functionally relevant OTUs that were positively correlated with MS were reduced by at least one of the probiotics, but each strain changed a distinct set of functionally relevant OTUs. LC and LR increased cecal acetate but did not affect circulating lipopolysaccharide-binding protein; in contrast, BA did not increase acetate but significantly decreased adipose and hepatic tumor necrosis factor-α gene expression. These results suggest that Lactobacillus and Bifidobacterium differentially attenuate obesity comorbidities in part through strain-specific impacts on MS-associated phylotypes of gut microbiota in mice.

Changes of the human gut microbiome induced by a fermented milk product.

The gut microbiota (GM) consists of resident commensals and transient microbes conveyed by the diet but little is known about the role of the latter on GM homeostasis. Here we show, by a conjunction of quantitative metagenomics, in silico genome reconstruction and metabolic modeling, that consumption of a fermented milk product containing dairy starters and Bifidobacterium animalis potentiates colonic short chain fatty acids production and decreases abundance of a pathobiont Bilophila wadsworthia compared to a milk product in subjects with irritable bowel syndrome (IBS, n = 28). The GM changes parallel improvement of IBS state, suggesting a role of the fermented milk bacteria in gut homeostasis. Our data challenge the view that microbes ingested with food have little impact on the human GM functioning and rather provide support for beneficial health effects.

Identification of restriction-modification systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT sequencing and associated methylome analysis.

Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in B. animalis subsp. lactis CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.

Gut microbiome composition and function in experimental colitis during active disease and treatment-induced remission.

Dysregulated immune responses to gut microbes are central to inflammatory bowel disease (IBD), and gut microbial activity can fuel chronic inflammation. Examining how IBD-directed therapies influence gut microbiomes may identify microbial community features integral to mitigating disease and maintaining health. However, IBD patients often receive multiple treatments during disease flares, confounding such analyses. Preclinical models of IBD with well-defined disease courses and opportunities for controlled treatment exposures provide a valuable solution. Here, we surveyed the gut microbiome of the T-bet(-/-) Rag2(-/-) mouse model of colitis during active disease and treatment-induced remission. Microbial features modified among these conditions included altered potential for carbohydrate and energy metabolism and bacterial pathogenesis, specifically cell motility and signal transduction pathways. We also observed an increased capacity for xenobiotics metabolism, including benzoate degradation, a pathway linking host adrenergic stress with enhanced bacterial virulence, and found decreased levels of fecal dopamine in active colitis. When transferred to gnotobiotic mice, gut microbiomes from mice with active disease versus treatment-induced remission elicited varying degrees of colitis. Thus, our study provides insight into specific microbial clades and pathways associated with health, active disease and treatment interventions in a mouse model of colitis.

Lactobacillus paracasei comparative genomics: towards species pan-genome definition and exploitation of diversity.

Lactobacillus paracasei is a member of the normal human and animal gut microbiota and is used extensively in the food industry in starter cultures for dairy products or as probiotics. With the development of low-cost, high-throughput sequencing techniques it has become feasible to sequence many different strains of one species and to determine its "pan-genome". We have sequenced the genomes of 34 different L. paracasei strains, and performed a comparative genomics analysis. We analysed genome synteny and content, focussing on the pan-genome, core genome and variable genome. Each genome was shown to contain around 2800-3100 protein-coding genes, and comparative analysis identified over 4200 ortholog groups that comprise the pan-genome of this species, of which about 1800 ortholog groups make up the conserved core. Several factors previously associated with host-microbe interactions such as pili, cell-envelope proteinase, hydrolases p40 and p75 or the capacity to produce short branched-chain fatty acids (bkd operon) are part of the L. paracasei core genome present in all analysed strains. The variome consists mainly of hypothetical proteins, phages, plasmids, transposon/conjugative elements, and known functions such as sugar metabolism, cell-surface proteins, transporters, CRISPR-associated proteins, and EPS biosynthesis proteins. An enormous variety and variability of sugar utilization gene cassettes were identified, with each strain harbouring between 25-53 cassettes, reflecting the high adaptability of L. paracasei to different niches. A phylogenomic tree was constructed based on total genome contents, and together with an analysis of horizontal gene transfer events we conclude that evolution of these L. paracasei strains is complex and not always related to niche adaptation. The results of this genome content comparison was used, together with high-throughput growth experiments on various carbohydrates, to perform gene-trait matching analysis, in order to link the distribution pattern of a specific phenotype to the presence/absence of specific sets of genes.

Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome.

Alterations in intestinal microbiota are associated with obesity and insulin resistance. We studied the effects of infusing intestinal microbiota from lean donors to male recipients with metabolic syndrome on the recipients' microbiota composition and glucose metabolism. Subjects were assigned randomly to groups that were given small intestinal infusions of allogenic or autologous microbiota. Six weeks after infusion of microbiota from lean donors, insulin sensitivity of recipients increased (median rate of glucose disappearance changed from 26.2 to 45.3 µmol/kg/min; P < .05) along with levels of butyrate-producing intestinal microbiota. Intestinal microbiota might be developed as therapeutic agents to increase insulin sensitivity in humans; www.trialregister.nl; registered at the Dutch Trial Register (NTR1776).

Microbial domestication signatures of Lactococcus lactis can be reproduced by experimental evolution.

Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and in dairy environments, and it is proposed that dairy strains originate from the plant niche. Here we investigate the adaptation of a L. lactis strain isolated from a plant to a dairy niche by propagating it for 1000 generations in milk. Two out of three independently evolved strains displayed significantly increased acidification rates and biomass yields in milk. Genome resequencing, revealed six, seven, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport and in the gene encoding MutL, which is involved in DNA mismatch repair. Two strains lost a conjugative transposon containing genes important in the plant niche but dispensable in milk. A plasmid carrying an extracellular protease was introduced by transformation. Although improving growth rate and growth yield significantly, the plasmid was rapidly lost. Comparative transcriptome and phenotypic analyses confirmed that major physiological changes associated with improved growth in milk relate to nitrogen metabolism and the loss or down-regulation of several pathways involved in the utilization of complex plant polymers. Reproducing the transition from the plant to the dairy niche through experimental evolution revealed several genome, transcriptome, and phenotype signatures that resemble those seen in strains isolated from either niche.

Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer.

In the past decade it has become clear that the lactic acid bacterium Lactobacillus plantarum occupies a diverse range of environmental niches and has an enormous diversity in phenotypic properties, metabolic capacity and industrial applications. In this review, we describe how genome sequencing, comparative genome hybridization and comparative genomics has provided insight into the underlying genomic diversity and versatility of L. plantarum. One of the main features appears to be genomic life-style islands consisting of numerous functional gene cassettes, in particular for carbohydrates utilization, which can be acquired, shuffled, substituted or deleted in response to niche requirements. In this sense, L. plantarum can be considered a "natural metabolic engineer".

Genome sequence of the probiotic strain Bifidobacterium animalis subsp. lactis CNCM I-2494.

Bifidobacterium animalis subsp. lactis CNCM I-2494 is part of a commercialized fermented dairy product with documented health benefits revealed by multiple randomized placebo-controlled clinical trials. Here we report the complete genome sequence of this strain, which has a circular genome of 1,943,113 bp with 1,660 open reading frames and 4 ribosomal operons.

Volatile compound fingerprinting of mixed-culture fermentations.

With the advent of the -omics era, classical technology platforms, such as hyphenated mass spectrometry, are currently undergoing a transformation toward high-throughput application. These novel platforms yield highly detailed metabolite profiles in large numbers of samples. Such profiles can be used as fingerprints for the accurate identification and classification of samples as well as for the study of effects of experimental conditions on the concentrations of specific metabolites. Challenges for the application of these methods lie in the acquisition of high-quality data, data normalization, and data mining. Here, a high-throughput fingerprinting approach based on analysis of headspace volatiles using ultrafast gas chromatography coupled to time of flight mass spectrometry (ultrafast GC/TOF-MS) was developed and evaluated for classification and screening purposes in food fermentation. GC-MS mass spectra of headspace samples of milk fermented by different mixed cultures of lactic acid bacteria (LAB) were collected and preprocessed in MetAlign, a dedicated software package for the preprocessing and comparison of liquid chromatography (LC)-MS and GC-MS data. The Random Forest algorithm was used to detect mass peaks that discriminated combinations of species or strains used in fermentations. Many of these mass peaks originated from key flavor compounds, indicating that the presence or absence of individual strains or combinations of strains significantly influenced the concentrations of these components. We demonstrate that the approach can be used for purposes like the selection of strains from collections based on flavor characteristics and the screening of (mixed) cultures for the presence or absence of strains. In addition, we show that strain-specific flavor characteristics can be traced back to genetic markers when comparative genome hybridization (CGH) data are available.

High-resolution amplified fragment length polymorphism typing of Lactococcus lactis strains enables identification of genetic markers for subspecies-related phenotypes.

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.