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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.

This work supports the massive presence of potential immunomodulatory peptides in the human gut metaproteome. These peptides were identified through the MAHMI database as potentially anti-inflammatory, and sixteen of them synthesized for characterize their mechanism of action. From them, peptide HM14 was encrypted in an extracellular protein produced by Bifidobacterium longum, a common member of the human microbiota, and displayed the highest anti-inflammatory capability. Molecular mechanism of action of HM14 pointed to a specific interaction between this immunomodulatory peptide and antigen presenting cells, which resulted in a higher formation of iTreg cells. Moreover, HM14 was effective in decreasing pro-inflammatory parameters in PBMCs isolated from a cohort of Crohn’s patients. Finally, non-targeted metabolomics confirmed the ability of HM14 to modulate the metabolic activity of PBMCs to fulfil its energy and biosynthetic requirements. Overall, our combined in silico/multiomics approach supports the human gut metaproteome as a source for immunomodulatory peptides.

Advances in the field of genome sequencing have enabled a comprehensive analysis and annotation of the dynamics of the protein inventory of cells. This has been proven particularly rewarding for microbial cells, for which the majority of proteins are already accessible to analysis through automatic metagenome annotation. The large-scale in silico screening of proteomes and metaproteomes is key to uncover bioactivities of translational, clinical and biotechnological interest, and to help assign functions to certain proteins, such as those predicted as hypothetical. This work introduces a new method for the prediction of the bioactivity potential of proteomes/metaproteomes, supporting the discovery of functionally relevant proteins based on prior knowledge. This methodology complements functional annotation enrichment methods by allowing the assignment of functions to proteins annotated as hypothetical/putative/uncharacterised, as well as and enabling the detection of specific bioactivities and the recovery of proteins from defined taxa. This work shows how the new method can be applied to screen proteome and metaproteome sets to obtain predictions of clinical or biotechnological interest based on reference datasets. Notably, with this methodology, the large information files obtained after DNA sequencing or protein identification experiments can be associated for translational purposes that, in cases such as antibiotic-resistance pathogens or foodborne diseases, may represent changes in how these important and global health burdens are approached in the clinical practice. Finally, the Sequence-based Expert-driven pRoteome bioactivity Prediction EnvironmENT, a public Web service implemented in Scala functional programming style, is introduced as means to ensure broad access to the method as well as to discuss main implementation issues, such as modularity, extensibility and interoperability.

[Background] Transcriptomics profiling aims to identify and quantify all transcripts present within a cell type or tissue at a particular state, and thus provide information on the genes expressed in specific experimental settings, differentiation or disease conditions. RNA-Seq technology is becoming the standard approach for such studies, but available analysis tools are often hard to install, configure and use by users without advanced bioinformatics skills. [Methods] Within reason, DEWE aims to make RNA-Seq analysis as easy for non-proficient users as for experienced bioinformaticians. DEWE supports two well-established and widely used differential expression analysis workflows: using Bowtie2 or HISAT2 for sequence alignment; and, both applying StringTie for quantification, and Ballgown and edgeR for differential expression analysis. Also, it enables the tailored execution of individual tools as well as helps with the management and visualisation of differential expression results. [Results] DEWE provides a user-friendly interface designed to reduce the learning curve of less knowledgeable users while enabling analysis customisation and software extension by advanced users. Docker technology helps overcome installation and configuration hurdles. In addition, DEWE produces high quality and publication-ready outputs in the form of tab-delimited files and figures, as well as helps researchers with further analyses, such as pathway enrichment analysis. [Conclusions] The abilities of DEWE are exemplified here by practical application to a comparative analysis of monocytes and monocyte-derived dendritic cells, a study of clinical relevance. DEWE installers and documentation are freely available at https://www.sing-group.org/dewe.

Probiotics are used to improve human health due to their capability to induce beneficial effects in gastrointestinal diseases and modulate the immune response. However, scarce information is known about the bacterial active compounds and the metabolic pathways of interaction with immune cells. In the present study, we analyzed the immune response elicited by surface-associated proteins, the whole surface extract (WSE) and the genomic DNA of three strains from the main representative probiotic species for human consumption, L. acidophilus DSM20079T, L. rhamnosus GG, B. longum NCIMB 8809, using an in vitro model of human PBMCs from healthy donors. Eighteen cytokines were quantified using Luminex Technology to understand the immune pathways modulate by bacterial extracts. Overall, the bacterial fractions elicited a different immune response in PBMCs, whereas the isolated DNA was able to induce a higher Th17-like response that could lead to a protective effect in the epithelial barrier function.

Probiotics are live microorganisms that confer a health benefit on the host when administered in adequate amounts. Toxicity induced by molecular oxygen significantly affects the viability of probiotic bacteria during biomass generation, product manufacturing and storage also constitute a major challenge for their inclusion in dairy products. Oxygen toxicity is particularly critical in the case of bifidobacteria, which are strict anaerobes. This mini-review aims at compiling the knowledge on the response mechanisms, mainly in this bacterial group, focused on counteracting the deleterious effects of oxygen on probiotic cells. The effect of other technological and microbiological ways to attenuate oxygen toxicity, such as milk deaeration, microencapsulation or co-culture is reviewed. Potential uses of the isolation of better adapted strains of probiotic Bifidobacterium strains, as well as the next generation probiotics such as Faecalibacterium prausnitzii, are discussed.

In this work, we have used a metataxonomic analysis to study the bacterial biodiversity in three types of commercial dressing and ingredients habitually used in table olive packaging. The bioinformatics analysis showed that salt samples (marine and spring) had the highest biodiversity indexes, followed by the aromatic herbs (thyme, oregano, and fennel) and finally by fermented dressing samples (garlic, pepper, and pepper paste). In salt samples, a significant high presence of Cytophagia, Sphingobacteria, Deltaproteobacteria and Halobacteria classes was observed. On the contrary, the presence of Bacilli class in fermented dressing samples was significantly higher compared to the other samples. Finally, the abundance of Gammaproteobacteria was statistically higher in the samples of herbs. At genus or family taxonomic level, Pseudomonas and diverse non-assigned genus of the Enterobacteriaceae family predominated in many samples of herbs. In fermented dressing samples, diverse genus belonging to the Enterobacteriaceae family appeared in a high relative abundance in pepper paste and garlic, and presence of Vibrio sp. was detected in pepper. However, Lactobacillus, Weisella, and Pediococcus were the predominant genera found in many of these fermented samples. Salinibacter was the most representative genus in both types of salt samples. Multivariate analysis (PCA) of the metataxonomic data sets discriminated well the samples belonging to the three different groups, showing a high proportion of sequences shared by samples-group. This survey reports for the first time the presence of new bacterial species not previously related with table olive processing, as well as the presence of others which could compromise the stability of olive packaging.

Peptides encrypted in the intestinal microbial-exoproteome mediate the host-microbiota crosstalk, which is disrupted in inflammatory bowel disease (IBD). Here, the MAHMI database was used for the identification of 20 novel intestinal bacterial peptides. Our results revealed that serum IgA levels directed towards the peptides, but not IgG, discriminated healthy controls from IBD patients. Indeed, they also differentiated patients with ulcerative colitis from Crohńs disease and, within them, patients with and without intestinal inflammation. All peptides were immunomodulatory as they changed the intestinal cytokine milieu following human lamina propria mononuclear cells culture (with/out LPS), revealing a Bifidobacterium longum subsp. longum peptide with the highest tolerogenic properties. Therefore, bacterial peptides encrypted in the human gut metaproteome may have utility as non-invasive biomarkers to aid on IBD diagnosis and monitoring. These peptides also display immunomodulatory effects on the intestinal mucosa revealing them as novel functional compounds for non-drug therapeutic strategies in IBD.

This work reports on a large-scale potential neuropeptide activity screening in human gut microbiomes deposited in public databases. In our experimental approach, the sequences of the bioactive peptides collected in the MAHMI database, mainly predicted as immunomodulatory or antitumoral, were crossed with those of the neuroactive/digestive peptides. From 91,325,790 potential bioactive peptides, only 581 returned a match when crossed against the 5949 neuroactive peptides from the NeuroPep database and the 15 digestive hormones. Relevant bacterial taxa, such as Ruminococcus sp., Clostridium sp. were found among the main producers of the matching sequences, and many of the matches corresponded to adiponectin and the hormone produced by adipocites, which is involved in glucose homeostasis. These results show, for the first time, the presence of potentially bioactive peptides produced by gut microbiota members over the nervous cells, most notably, peptides with already predicted immunomodulatory or anti-inflammatory activity. Classical (Lactobacillus sp.) and next-generation (Faecalibacterium sp.) probiotics are shown to produce these peptides, which are proposed as a potential mechanism of action of psychobiotics. Our previous experimental results showed that many of these peptides were active when incubated with immune cells, such as dendritic cells, so their effect over the nervous system innervating the gut mucosa holds significant potential and should be explored.

Bifidobacteria are gut commensal microorganisms belonging to the Actinobacteria group. Some specific strains of Bifidobacterium animalis subsp. lactis are used in functional foods as they are able to exert health-promoting effects in the human host. Due to the limited genetic variability within this subspecies, it is sometimes difficult for a manufacturer to properly track its strain once included in dairy products or functional foods. In this paper, we present a peptidome-based analysis in which the proteomes of a set of B. animalis subsp. lactis strains were digested in silico with human gut endopeptidases. The molecular masses were compared along all the strains to detect strain-specific peptides. These peptides may be interesting towards the development of methodologies for strain identification in the final product.

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Many strains exert their beneficial effects after transiently colonizing the human gut, where they interact with the rest of the intestinal microorganisms and with the host mucosa. Indeed the human gut harbours a huge number of microorganisms also known as gut microbiota. Imbalances in the relative abundances of the individual components of the gut microbiota may determine the health status of the host and alterations in specific groups have been related to different diseases and metabolic disorders. Proteomics provide a set of high-throughput methodologies for protein identification that are extremely useful for studying probiotic functionality and helping in the assessment of specific health-promoting activities, such as their immunomodulatory activity, the intestinal colonization processes, and the crosstalk mechanisms with the host. Furthermore, proteomics have been used to identify markers of technological performance and stress adaptation, which helps to predict traits such as behaviour into food matrices and ability to survive passage through the gastrointestinal tract. The aim of this review is to compile studies in which proteomics have been used to assess probiotic functionality and to identify molecular players supporting their mechanisms of action. Significance Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Molecular basis underlying the functional properties of probiotic bacteria responsible for the health promoting effects have been in the background for many years. Breakthrough of omics technologies in the probiotic and microbiota fields has had a very relevant impact in the elucidation of probiotic mechanisms and in the procedures to select these microorganisms, based on solid scientific evidence. It is unquestionable that, in the near future, the evolution of proteomi

Objectives To assess the establishment of the intestinal microbiota in very low birthweight preterm infants and to evaluate the impact of perinatal factors, such as delivery mode and perinatal antibiotics. Study design We used 16S ribosomal RNA gene sequence-based microbiota analysis and quantitative polymerase chain reaction to evaluate the establishment of the intestinal microbiota. We also evaluated factors affecting the microbiota, during the first 3 months of life in preterm infants (n = 27) compared with full-term babies (n = 13). Results Immaturity affects the microbiota as indicated by a reduced percentage of the family Bacteroidaceae during the first months of life and by a higher initial percentage of Lactobacillaceae in preterm infants compared with full term infants. Perinatal antibiotics, including intrapartum antimicrobial prophylaxis, affects the gut microbiota, as indicated by increased Enterobacteriaceae family organisms in the infants. Conclusions Prematurity and perinatal antibiotic administration strongly affect the initial establishment of microbiota with potential consequences for later health. © 2015 Elsevier Inc.

Probiotics are live microorganisms that are able to confer health benefits on the host, in most cases through their interaction with the local gastrointestinal environment. Traditionally, they have been selected for their technological parameters, without following a scientific rationale for specific applications. However, high-throughput methodologies have allowed the scientific community to establish novel probiotic markers, potentially helping to understand the probiotic effect, as well as traits related to the robustness of these microorganisms. These markers could be used for strain selection and included among the selection criteria for new probiotics. Furthermore, strategies directed at enhancing the functionality of the already available strains have also been developed. © 2012 Elsevier Ltd.

The characterisation of 24 lactic acid bacteria (LAB) isolates from Nigerian traditional fermented dairy foods, including some cow's intestine isolates, was conducted in order to select isolates for potential use as probiotics. LAB isolates were identified by partial sequencing the 16S rRNA gene as belonging to the species Lactobacillus paracasei, Lactobacillus brevis and mainly Weissella confusa. At the end of a characterisation process, 2 L. paracasei and 2 W. confusa isolates were selected, and their resistance to a simulated gastrointestinal digestion and their ability to adhere to eukaryotic cell lines were assessed. The survival to the simulated gastrointestinal passage was higher when bacterial suspensions were made in skimmed milk (2.0 ± 0.8 log units reduction) or at the simulated gastric juice pH 3 (2.7 ± 0.9 log units reduction) than at pH 2.0 (5.5 ± 0.7 log units reduction). Adhesion of LAB to both intestinal and vaginal epithelial models was comparable or higher than that of the reference Lactobacillus rhamnosus GG. However, some of the isolates increased the adhesion of the pathogen Escherichia coli LMG2092 to HT-29 and HeLa monolayers. Overall, isolates L. paracasei UI14 and W. confusa UI7 are good candidates for further studying potential benefits that support their use as probiotics. This is one of the few articles reporting the characterisation and the probiotic potential of Weissella, although more studies are needed in order to establish their safety for potential probiotic applications. © 2011 Elsevier B.V.

Staphylococcus aureus is an important pathogenic microorganism that has been associated with serious infection problems in different fields, from food to clinic. In the present study, we have taken into account that the main reservoirs of this microorganism are the human body and some parts of food processing plants, which have normal temperatures of around 37 and 25 °C, respectively. It can be expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food matrices. Since temperature abuse for foods occurs at approximately 12 °C, it is expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food. For this reason, we have performed a proteomic comparison between exponential- and stationary-phase cultures of S. aureus CECT 976 acclimated to 12 °C after growing at 25 °C or 37 °C. The analysis led to the identification of two different protein patterns associated with cold acclimation, denominated pattern A and pattern B. The first was characteristic of cultures at stationary phase of growth, grown at 25 °C and acclimated to 12 °C. The second appeared in the rest of experimental cases. Pattern A was distinguished by the presence of glycolytic proteins, whereas pattern B was differentiated by the presence of general stress and regulatory proteins. Pattern A was related through physiological experiments with a cross-resistance to acid pH, whereas pattern B conferred resistance to nisin. This prompted us to conclude that both molecular strategies could be valid, in vivo, for the process of acclimation of S. aureus to cold temperatures.

Progressive adaptation to bile might render some lactobacilli able to withstand physiological bile salt concentrations. In this work, the adaptation to bile was evaluated on previously isolated dairy strains of Lactobacillus delbrueckii subsp. lactis 200 and L. delbrueckii subsp. lactis 200+, a strain derived thereof with stable bile-resistant phenotype. The adaptation to bile was obtained by comparing cytosolic proteomes of both strains grown in the presence or absence of bile. Proteomics were complemented with physiological studies on both strains focusing on glycolytic end-products, the ability to adhere to the human intestinal epithelial cell line HT29-MTX and survival to simulated gastrointestinal conditions. Protein pattern comparison of strains grown with and without bile allowed us to identify 9 different proteins whose production was regulated by bile in both strains, and 17 proteins that showed differences in their levels between the parental and the bile-resistant derivative. These included general stress response chaperones, proteins involved in transcription and translation, in peptidoglycan/exopolysaccharide biosynthesis, in the lipid and nucleotide metabolism and several glycolytic and pyruvate catabolism enzymes. Differences in the level of metabolic end-products of the sugar catabolism were found between the strains 200 and 200+. A decrease in the adhesion of both strains to the intestinal cell line was detected in the presence of bile. In simulated gastric and intestinal juices, a protective effect was exerted by milk improving the survival of both microorganisms. These results indicate that bile tolerance in L. delbrueckii subsp. lactis involves several mechanisms responding to the deleterious impact of bile salts on bacterial physiology. © 2010 Elsevier B.V.

The technological and probiotic properties of the bile-adapted strain Bifidobacterium animalis subsp. lactis 4549dOx have been studied in comparison with its parental strain IPLA4549. The survival of the bile-adapted derivative to the simulated gastrointestinal transit was significantly higher than that of the parental strain, except in the presence of 10% skimmed milk. The adherence of strain 4549dOx to the intestinal cell line HT29-MTX almost duplicated that of strain IPLA4549. However, both strains had similar behaviour throughout the shelf-life of fermented milk. In conclusion, the bile-adapted strain 4549dOx presented advantages for its inclusion in dairy probiotic foods. © 2010 Elsevier Ltd.