A methyl esterase from Bifidobacterium longum subsp. longum reshapes the prebiotic properties of apple pectin by triggering differential modulatory capacity in faecal cultures.

Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity of gut bacterial communities to cooperatively metabolize structural variants of this substrate remains largely unknown. Herein, the characterization of a pectin methylesterase, BpeM, from Bifidobacterium longum subsp. longum, is reported. The purified enzyme was able to remove methyl groups from highly methoxylated apple pectin, and the mathematical modelling of its activity enabled to tightly control the reaction conditions to achieve predefined final degrees of methyl-esterification in the resultant pectin. Demethylated pectin, generated by BpeM, exhibited differential fermentation patterns by gut microbial communities in in vitro mixed faecal cultures, promoting a stronger increase of bacterial genera associated with beneficial effects including Lactobacillus, Bifidobacterium and Collinsella. Our findings demonstrate that controlled pectin demethylation by the action of a B. longum esterase selectively modifies its prebiotic fermentation pattern, producing substrates that promote targeted bacterial groups more efficiently. This opens new possibilities to exploit biotechnological applications of enzymes from gut commensals to programme prebiotic properties.

The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits.

BACKGROUND: Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. RESULTS: Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and ß-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. CONCLUSION: Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract.

Dynamics of Gut Microbiota and Short-Chain Fatty Acids during a Cycling Grand Tour Are Related to Exercise Performance and Modulated by Dietary Intake.

BACKGROUND: Regular exercise has been described to modify both the diversity and the relative abundance of certain bacterial taxa. To our knowledge, the effect of a cycling stage race, which entails extreme physiological and metabolic demands, on the gut microbiota composition and its metabolic activity has not been analysed. OBJECTIVE: The aim of this cohort study was to analyse the dynamics of faecal microbiota composition and short-chain fatty acids (SCFAs) content of professional cyclists over a Grand Tour and their relationship with performance and dietary intake. METHODS: 16 professional cyclists competing in La Vuelta 2019 were recruited. Faecal samples were collected at four time points: the day before the first stage (A); after 9 stages (B); after 15 stages (C); and on the last stage (D). Faecal microbiota populations and SCFA content were analysed using 16S rRNA sequencing and gas chromatography, respectively. A principal component analysis (PCA) followed by Generalised Estimating Equation (GEE) models were carried out to explore the dynamics of microbiota and SCFAs and their relationship with performance. RESULTS: Bifidobacteriaceae, Coriobacteriaceae, Erysipelotrichaceae, and Sutterellaceae dynamics showed a strong final performance predictive value (r = 0.83, ranking, and r = 0.81, accumulated time). Positive correlations were observed between Coriobacteriaceae with acetate (r = 0.530) and isovalerate (r = 0.664) and between Bifidobacteriaceae with isobutyrate (r = 0.682). No relationship was observed between SCFAs and performance. The abundance of Erysipelotrichaceae at the beginning of La Vuelta was directly related to the previous intake of complex-carbohydrate-rich foods (r = 0.956), while during the competition, the abundance of Bifidobacteriaceae was negatively affected by the intake of simple carbohydrates from supplements (r = -0.650). CONCLUSIONS: An ecological perspective represents more realistically the relationship between gut microbiota composition and performance compared to single-taxon approaches. The composition and periodisation of diet and supplementation during a Grand Tour, particularly carbohydrates, could be designed to modulate gut microbiota composition to allow better performance.

Study of the intestinal microbiota composition and the effect of treatment with intensive chemotherapy in patients recovered from acute leukemia.

A dataset comprising metagenomes of outpatients (n = 28) with acute leukemia (AL) and healthy controls (n = 14) was analysed to investigate the associations between gut microbiota composition and metabolic activity and AL. According to the results obtained, no significant differences in the microbial diversity between AL outpatients and healthy controls were found. However, significant differences in the abundance of specific microbial clades of healthy controls and AL outpatients were found. We found some differences at taxa level. The relative abundance of Enterobacteriaceae, Prevotellaceae and Rikenellaceae was increased in AL outpatients, while Bacteirodaceae, Bifidobacteriaceae and Lachnospiraceae was decreased. Interestingly, the abundances of several taxa including Bacteroides and Faecalibacterium species showed variations based on recovery time from the last cycle of chemotherapy. Functional annotation of metagenome-assembled genomes (MAGs) revealed the presence of functional domains corresponding to therapeutic enzymes including L-asparaginase in a wide range of genera including Prevotella, Ruminococcus, Faecalibacterium, Alistipes, Akkermansia. Metabolic network modelling revealed potential symbiotic relationships between Veillonella parvula and Levyella massiliensis and several species found in the microbiota of AL outpatients. These results may contribute to develop strategies for the recovery of microbiota composition profiles in the treatment of patients with AL.

Gut microbiota modulation and effects of a diet enriched in apple pomace on inflammation in a DSS-induced colitis mouse model.

Certain types of soluble dietary fibre, such as pectin and pectic oligosaccharides from different sources, have demonstrated protective effects against inflammation in DSS-induced colitis mouse models. In this work, we have evaluated the impact of a diet enriched in apple pomace (AP-diet), an agricultural by-product with a significant content of pectin and that previously demonstrated prebiotic properties in human fecal batch fermentation models, on the gut microbiota composition, intestinal damage and inflammation markers in a DSS-induced colitis model. We found that the apple pomace enriched diet (AP-diet), providing a significant amount of pectin with demonstrated prebiotic properties, was associated with a slower increase in the disease activity index, translating into better clinical symptomatology of the animals. Histological damage scoring confirmed less severe damage in those animals receiving an AP-diet before and during the DSS administration period. Some serum inflammatory markers, such as TNFα, also demonstrated lower levels in the group receiving the AP-diet, compared to the control diet. AP-diet administration is also associated with the modulation of key taxa in the colonic microbiota of animals, such as some Lachnospiraceae genera and Ruminococcus species, including commensal short chain fatty acid producers that could play a role in attenuating inflammation at the intestinal level.

Gut microbiota and inflammatory mediators differentiate IgE mediated and non-IgE mediated cases of cow's milk protein at diagnosis.

OBJECTIVE: Analyze fecal and blood samples at point of diagnosis in IgE mediated cow's milk protein allergy (CMPA) and non-IgE mediated (NIM)-CMPA patients to look for potential new biomarkers. PATIENTS AND METHODS: Fourteen patients with IgE mediated CMPA and 13 with NIM-CMPA were recruited in three hospitals in the north of Spain, and were compared with 25 infants from a control group of the same age range. To characterize intestinal microbiota, 16S rDNA gene and internal transcribed spacer amplicons of bifidobacteria were sequenced with Illumina technology. Fatty acids were analyzed by gas chromatography, meanwhile intestinal inflammation markers were quantified by enzyme-linked immunosorbent assay and a multiplex system. Immunological analysis of blood was performed by flow cytometry. RESULTS: The fecal results obtained in the NIM-CMPA group stand out. Among them, a significant reduction in the abundance of Bifidobacteriaceae and Bifidobacterium sequences with respect to controls was observed. Bifidobacterial species were also different, highlighting the lower abundance of Bifidobacterium breve sequences. Fecal calprotectin levels were found to be significantly elevated in relation to IgE mediated patients. Also, a higher excretion of IL-10 and a lower excretion of IL-1ra and platelet derived growth factor-BB was found in NIM-CMPA patients. CONCLUSIONS: The differential fecal parameters found in NIM-CMPA patients could be useful in the diagnosis of NIM food allergy to CM proteins.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Mouse intestinal microbiome modulation by oral administration of a GABA-producing Bifidobacterium adolescentis strain.

IMPORTANCE: The gut microbiome-brain communication signaling has emerged in recent years as a novel target for intervention with the potential to ameliorate some conditions associated with the central nervous system. Hence, probiotics with capacity to produce neurotransmitters, for instance, have come up as appealing alternatives to treat disorders associated with disbalanced neurotransmitters. Herein, we further deep into the effects of administering a gamma-aminobutyric acid (GABA)-producing Bifidobacterium strain, previously demonstrated to contribute to reduce serum glutamate levels, in the gut microbiome composition and metabolic activity in a mouse model. Our results demonstrate that the GABA-producing strain administration results in a specific pattern of gut microbiota modulation, different from the one observed in animals receiving non-GABA-producing strains. This opens new avenues to delineate the specific mechanisms by which IPLA60004 administration contributes to reducing serum glutamate levels and to ascertain whether this effect could exert health benefits in patients of diseases associated with high-glutamate serum concentrations.

Next-generation sequencing of the athletic gut microbiota: a systematic review.

Aim: There is growing evidence that physical activity modulates gut microbiota composition through complex interactions between diet and microbial species. On the other hand, next-generation sequencing techniques include shotgun metagenomics and 16S amplicon sequencing. These methodologies allow a comprehensive characterisation of microbial communities of athletes from different disciplines as well as non-professional players and sedentary adults exposed to training. This systematic review summarises recent applications of next-generation sequencing to characterise the athletic gut microbiome. Methods: A systematic review of microbiome research was performed to determine the association of microbiota composition profiles with sports performance. Results: Bibliographic analysis revealed the importance of a novel research trend aiming at deciphering the associations between individual microbial species and sports performance. In addition, literature review highlighted the role of butyrate-producing bacteria such as Anaerostipes hadrus, Clostridium bolteae, Faecalibacterium prausnitzii, Roseburia hominis and unidentified species belonging to Clostridiales, Lachnospiraceae and Subdoligranulum species in gut health and sports performance across several disciplines. Interestingly, metabolic activities of Prevotella copri and Veillonella atypica involved in branched amino acid and lactate metabolism may contribute to reducing muscular fatigue. Other microbial metabolic pathways of interest involved in carbohydrate metabolism showed increased proportions in athletes´ metagenomes. Conclusion: Future research will aim at developing personalised nutrition interventions to modulate key species associated with certain components of exercise.

Metabolic response of intestinal microbiota to guar gum consumption.

Background: Guar gum is used extensively as a thickening agent in food, but it remains uncertain whether and to what extent it is fermented by colonic microbiota and whether it has microbiota modulatory properties. Aim: To determine the metabolic response of intestinal microbiota to guar gum consumption, specifically, the extent of initial fermentation and subsequent adaptation. Methods: Single-center, single arm, open label, proof-of-concept study testing the effect of guar gum on microbiota metabolism and adaptation. Healthy male subjects (n = 12) were administered gum guar (8 g/day) for 18 days. Outcomes were measured before, at initial and late administration: (a) anal gas evacuations (number/day); (b) digestive sensations (daily scales); and (c) fecal gut microbiota taxonomy and metabolic functions by shotgun sequencing. Results: At initial consumption, guar gum induced a transient increase in anal gas evacuations and digestive sensations; gas evacuation completely reverted upon continuous administration, whereas sensations reverted only in part. Guar gum induced moderate changes in human microbiota composition at both taxonomic and functional levels. Positive associations between effects on microbiota (proliferation of Agathobaculum butyriciproducens and Lachnospira pectinoschiza) and hedonic sensations were detected. Conclusion: Guar gum is metabolized by intestinal microbiota, and, upon continuous consumption, induces a selective adaptation of microbial taxonomy and function. These data highlight the potential interest of guar gum for novel prebiotic ingredient formulation.

Production of GABA in milk fermented by Bifidobacterium adolescentis strains selected on the bases of their technological and gastrointestinal performance.

There is an increasing interest in producing foods enriched in gamma-aminobutyric acid (GABA), due to their purported health promoting attributes. GABA is the main inhibitor neurotransmitter of the central nervous system, and several microbial species are capable to produce it through decarboxylation of glutamate. Among them, several lactic acid bacteria species have been previously investigated as an appealing alternative to produce GABA enriched foods via microbial fermentation. In this work we report for the first time an investigation into the possibility of utilizing high GABA-producing Bifidobacterium adolescentis strains as a mean to produce fermented probiotic milks naturally enriched in GABA. To this end, in silico and in vitro analyses were conducted in a collection of GABA-producing B. adolescentis strains, with the main goal to scrutinize their metabolic and safety traits, including antibiotic resistance patterns, as well as their technological robustness and performance to survive a simulated gastrointestinal passage. One of the strains, IPLA60004, exhibited better survival to lyophilization and cold storage (for up to 4 weeks at 4 °C), as well as survival to gastrointestinal passage, as compared to the other strains under investigation. Besides, the elaboration of milk drinks fermented with this strain, yielded products with the highest GABA concentration and viable bifidobacterial cell counts, achieving conversion rates of the precursor, monosodium glutamate (GMS), up to 70 %. To our knowledge, this is the first report on the elaboration of GABA enriched milks through fermentation with B. adolescentis.

Gut epithelial barrier dysfunction in lupus triggers a differential humoral response against gut commensals.

Introduction: Systemic lupus erythematosus is an autoimmune disease with multisystemic involvement including intestinal inflammation. Lupus-associated intestinal inflammation may alter the mucosal barrier where millions of commensals have a dynamic and selective interaction with the host immune system. Here, we investigated the consequences of the intestinal inflammation in a TLR7-mediated lupus model. Methods: IgA humoral and cellular response in the gut was measured. The barrier function of the gut epithelial layer was characterised. Also, microbiota composition in the fecal matter was analysed as well as the systemic humoral response to differential commensals. Results: The lupus-associated intestinal inflammation modifies the IgA+ B cell response in the gut-associated lymphoid tissue in association with dysbiosis. Intestinal inflammation alters the tight junction protein distribution in the epithelial barrier, which correlated with increased permeability of the intestinal barrier and changes in the microbiota composition. This permeability resulted in a differential humoral response against intestinal commensals. Discussion: Lupus development can cause alterations in microbiota composition, allowing specific species to colonize only the lupus gut. Eventually, these alterations and the changes in gut permeability induced by intestinal inflammation could lead to bacterial translocation.

Orally Administered Bifidobacterium adolescentis Diminishes Serum Glutamate Concentration in Mice.

Several studies have described the contribution of glutamate-transforming microbiota to the development of chronic ailments. For instance, the blood concentration of glutamate is higher in some patients with fibromyalgia, chronic fatigue, and pain. Taking advantage of a naturally occurring strain of Bifidobacterium that is able to transform glutamate in γ-aminobutyric caid (GABA), B. adolescentis IPLA60004, we designed a placebo-controlled intervention to test if the presence of this GABA-producing bifidobacteria in mice was able to impact the concentration of glutamate in the blood in comparison with the administration of other strain of the same species lacking the genes of the glutamate decarboxylase (gad) cluster. Animals were fed every day with 8 log CFU of bacteria in a sterilized milk vehicle for 14 days. Samples from feces and blood were collected during this period, and afterwards animals were sacrificed, tissues were taken from different organs, and the levels of different metabolites were analyzed by ultrahigh-performance liquid chromatography coupled to mass spectrometry. The results showed that both bacterial strains orally administered survived in the fecal content, and animals fed B. adolescentis IPLA60004 showed a significant reduction of their glutamate serum concentration, while a nonsignificant decrease was observed for animals fed a reference strain, B. adolescentis LGM10502. The variations observed in GABA were influenced by the gender of the animals, and no significant changes were observed in different tissues of the brain. These results suggest that orally administered GABA-producing probiotics could reduce the glutamate concentration in blood, opening a case for a clinical trial study in chronic disease patients. IMPORTANCE This work presents the results of a trial using mice as a model that were fed with a bacterial strain of the species B. adolescentis, which possesses different active genes capable of degrading glutamate and converting it into GABA. Indeed, the bacterium is able to survive the passage through the gastric tract and, more importantly, the animals reduce over time the concentration of glutamate in their blood. The importance of this result lies in the fact that several chronic ailments, such as fibromyalgia, are characterized by an increase in glutamate. Our results indicate that an oral diet with this probiotic-type bacteria could reduce the concentration of glutamate and, therefore, reduce the symptoms associated with the excess of this neurotransmitter.

Targeted depletion of pks+ bacteria from a fecal microbiota using specific antibodies.

The pks island is one of the most prevalent pathogenicity islands among the Escherichia coli strains that colonize the colon of colorectal carcinoma (CRC) patients. This pathogenic island encodes the production of a nonribosomal polyketide-peptide named colibactin, which induces double-strand breaks in DNA molecules. Detection or even depletion of this pks-producing bacteria could help to understand the role of these strains in the context of CRC. In this work, we performed a large-scale in silico screening of the pks cluster in more than 6,000 isolates of E. coli. The results obtained reveal that not all the pks-detected strains could produce a functional genotoxin and, using antibodies against pks-specific peptides from surface cell proteins, a methodology for detection and depletion of pks+ bacteria in gut microbiotas was proposed. With our method, we were able to deplete a human gut microbiota of this pks+ strains, opening the door to strain-directed microbiota modification and intervention studies that allow us to understand the relation between these genotoxic strains and some gastrointestinal diseases. IMPORTANCE The human gut microbiome has also been hypothesized to play a crucial role in the development and progression of colorectal carcinoma (CRC). Between the microorganisms of this community, the Escherichia coli strains carrying the pks genomic island were shown to be capable of promoting colon tumorigenesis in a colorectal cancer mouse model, and their presence seems to be directly related to a distinct mutational signature in patients suffering CRC. This work proposes a novel method for the detection and depletion of pks-carrying bacteria in human gut microbiotas. In contrast to methods based on probes, this methodology allows the depletion of low-abundance bacterial strains maintaining the viability of both targeted and non-targeted fractions of the microbiota, allowing the study of the contribution of these pks-carrying strains to different diseases, such as CRC, and their role in other physiological, metabolic or immune processes.

Arabinoxylan-based substrate preferences and predicted metabolic properties of Bifidobacterium longum subspecies as a basis to design differential media.

Arabinoxylan (AX) and arabinoxylo-oligosaccharides (AXOS) derived therefrom are emergent prebiotics with promising health promoting properties, likely linked to its capacity to foster beneficial species in the human gut. Bifidobacteria appear to be one taxa that is frequently promoted following AX or AXOS consumption, and that is known to establish metabolic cross-feeding networks with other beneficial commensal species. Therefore, probiotic bifidobacteria with the capability to metabolize AX-derived prebiotics represent interesting candidates to develop novel probiotic and synbiotic combinations with AX-based prebiotics. In this work we have deepen into the metabolic capabilities of bifidobacteria related to AX and AXOS metabolization through a combination of in silico an in vitro tools. Both approaches revealed that Bifidobacterium longum and, particularly, B. longum subsp. longum, appears as the better equipped to metabolize complex AX substrates, although other related subspecies such as B. longum subsp. infantis, also hold some machinery related to AXOS metabolization. This correlates to the growth profiles exhibited by representative strains of both subspecies in AX or AXOS enriched media. Based on these results, we formulated a differential carbohydrate free medium (CFM) supplemented with a combination of AX and AXOS that enabled to recover a wide diversity of Bifidobacterium species from complex fecal samples, while allowing easy discrimination of AX metabolising strains by the appearance of a precipitation halo. This new media represent an appealing alternative to isolate novel probiotic bifidobacteria, rapidly discriminating their capacity to metabolize structurally complex AX-derived prebiotics. This can be convenient to assist formulation of novel functional foods and supplements, including bifidobacterial species with capacity to metabolize AX-derived prebiotic ingredients.

Towards the isolation of more robust next generation probiotics: The first aerotolerant Bifidobacterium bifidum strain.

This work reports on the first described aerotolerant Bifidobacterium bifidum strain, Bifidobacterium bifidum IPLA60003, which has the ability to form colonies on the surface of agar plates under aerobic conditions, a weird phenotype that to our knowledge has never been observed in B. bifidum. The strain IPLA60003 was generated after random UV mutagenesis from an intestinal isolate. It incorporates 26 single nucleotide polymorphisms that activate the expression of native oxidative-defense mechanisms such as the alkyl hydroxyperoxide reductase, the glycolytic pathway and several genes coding for enzymes involved in redox reactions. In the present work, we discuss the molecular mechanisms underlying the aerotolerance phenotype of B. bifidum IPLA60003, which will open new strategies for the selection and inclusion of probiotic gut strains and next generation probiotics into functional foods.

Evaluation of a Shotgun Metagenomics Approach for Detection of ESBL- and/or Carbapenemase-Producing Enterobacterales in Culture Negative Patients Recovered from Acute Leukemia.

Patients diagnosed with acute leukemia (AL) have a weakened immune system. Infections acquired by these patients are cause for concern and especially worrisome when Gram-negative multidrug-resistant (MDR) bacteria are involved, as they are difficult to treat, especially in the case of ESBL- and/or carbapenemase-producing Enterobacterales. Culture-based approaches have been relied on over the past decades as the method of choice for the early detection of gut colonization by MDR Gram-negative bacteria. However, various studies have indicated its limited sensitivity, underlining the need for new screening procedures in onco-hematological patients. Here, we evaluated a shotgun metagenomics approach to detect ESBL- and/or carbapenemase-producing Enterobacterales in the gut of 28 patients who had recovered from AL, which were previously colonized by these bacteria but cured at the time of sampling, as judged by culture-based methods. No ESBL or carbapenemase determinants were detected among the many resistance genes found by the metagenomics approach, supporting that patients were truly decolonized, with considerable consequences for their future clinical management. Due to the relatively low number of patients available for the present investigation, further studies should be conducted to support the utility and applicability of metagenomics for the routine screening of MDR bacteria in onco-hematological patients.

Immunomagnetic Capture of Faecalibacterium prausnitzii Selectively Modifies the Fecal Microbiota and Its Immunomodulatory Profile.

Faecalibacterium represents one of the most abundant bacterial groups in the human intestinal microbiota of healthy adults and can represent more than 10% of the total bacterial population, Faecalibacterium prausnitzii being the only recognized species up to the past year. Reduction in the abundance of F. prausnitzii in the human gut has been linked to several human disorders, such as Crohn's disease. In this study, we developed a strategy to modify the relative abundance of F. prausnitzii in fecal microbiotas as a means of evaluating its contribution to the immunomodulatory effect of intestinal microbiotas with different F. prausnitzii contents using a peripheral blood mononuclear cell (PBMC) model. We used a polyclonal antibody against the surface of F. prausnitzii M21 to capture the bacterium from synthetic and human fecal microbiotas using immunoseparation techniques. As a proof-of-principle study, the levels of immunomodulation exerted by microbiotas of healthy donors (HDs) with different relative abundances of F. prausnitzii, achieved with the above-mentioned immunoseparation technique, were evaluated in a PBMC model. For this purpose, PBMCs were cocultivated with the modified microbiotas or a pure culture of F. prausnitzii and, subsequently, the microbiota of Crohn's donors was added to the coculture. The cytokine concentration was determined, showing that our experimental model supports the anti-inflammatory effects of this bacterium. IMPORTANCE There is increasing interest in deciphering the contribution of gut microbiota species to health and disease amelioration. The approach proposed herein provides a novel and affordable strategy to probe deeply into microbiota-host interactions by strategically modifying the relative abundance of specific gut microbes, hence facilitating the study of their contribution to a given trait of the microbiota.

Convergence of flow cytometry and bacteriology. Current and future applications: a focus on food and clinical microbiology.

Since its development in the 1960s, flow cytometry (FCM) was quickly revealed a powerful tool to analyse cell populations in medical studies, yet, for many years, was almost exclusively used to analyse eukaryotic cells. Instrument and methodological limitations to distinguish genuine bacterial signals from the background, among other limitations, have hampered FCM applications in bacteriology. In recent years, thanks to the continuous development of FCM instruments and methods with a higher discriminatory capacity to detect low-size particles, FCM has emerged as an appealing technique to advance the study of microbes, with important applications in research, clinical and industrial settings. The capacity to rapidly enumerate and classify individual bacterial cells based on viability facilitates the monitoring of bacterial presence in foodstuffs or clinical samples, reducing the time needed to detect contamination or infectious processes. Besides, FCM has stood out as a valuable tool to advance the study of complex microbial communities, or microbiomes, that are very relevant in the context of human health, as well as to understand the interaction of bacterial and host cells. This review highlights current developments in, and future applications of, FCM in bacteriology, with a focus on those related to food and clinical microbiology.