The anticancer activity of bovine lactoferrin is reduced by deglycosylation and it follows a different pathway in cervix and colon cancer cells.

Bovine lactoferrin (bLF) is a glycosylated protein with purported beneficial properties. The aim of this work was to determine the role of bLF glycosylation in the adhesion, internalization, and growth inhibition of cancer cells. The viability of cervix (HeLa) and colon (Caco-2) cancer cells (MTT assay and epifluorescence microscopy) was inhibited by bLF, while deglycosylated bLF (bLFdeg) had no effect. Adhesion to cell surfaces was quantified by immunofluorescence assay and showed that bLF was able to bind more efficiently to both cell lines than bLFdeg. Microscopic observations indicated that bLF glycosylation favored bLF binding to epithelial cells and that it was endocytosed through caveolin-1-mediated internalization. In addition, the mechanism of action of bLF on cancer cell proliferation was investigated by determining the amount of phosphorylated intermediates of signaling pathways such as epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) and protein kinase B (known as Akt). Chemoluminescence immunoassay of phosphorylated intermediates showed that bLF inhibited Akt phosphorylation, consistent with its growth inhibiting activity. This assay also indicated that the bLF receptor/signaling pathways may be different in the two cell lines, Caco-2 and HeLa. This work confirmed the effect of glycosylated bLF in inhibiting cancer cell growth and that glycosylation is required for optimal surface adhesion, internalization, and inhibition of the ERK/Akt pathway of cell proliferation through glycosylated cell surface receptors.

Bacterial extracellular vesicles and associated functional proteins in fermented dairy products with Lacticaseibacillus paracasei.

Cells of all kingdoms produce extracellular vesicles (EVs); hence, they are present in most environments and body fluids. Lacticaseibacillus paracasei produces EVs that have attached biologically active proteins (P40 and P75). In this study, EV and functional proteins were found in five different commercial dairy-fermented products carrying L. paracasei. Strains present in those products were isolated, and with one exception, all produced small EVs (24-47 d.nm) carrying P40 and P75. In order to winnow bacterial EV from milk EV, products were subjected to centrifugal fractionation at 15,000 × g (15 K), 33,000 × g (33 K), and 100,000 × g (100 K). P75 was present in all supernatants and pellets, but P40 was only found in two products bound to the 15 and 33 K pellets, and 16S rDNA of L. paracasei could be amplified from all 100 K EVs, indicating the presence of L. paracasei EV. To investigate the interactions of bacterial EV and proteins with milk EV, L. paracasei BL23 EV was added to three commercial UHT milk products. Small-size vesicles (50-60 d.nm) similar to L. paracasei BL23 EV were found in samples from 100 K centrifugations, but intriguingly, P40 and P75 were bound to EV in 15 and 33 K pellets, containing bovine milk EV of larger size (200-300 d.nm). Sequencing 16S rDNA bands amplified from EV evidenced the presence of bacterial EVs of diverse origins in milk and fermented products. Furthermore, L. paracasei 16S rDNA could be amplified with species-specific primers from all samples, showing the presence of L. paracasei EV in all EV fractions (15, 33, and 100 K), suggesting that these bacterial EVs possibly aggregate and are co-isolated with EV from milk. P40 and P75 proteins would be interacting with specific populations of milk EV (15 and 33 K) because they were detected bound to them in fermented products and milk, and this possibly forced the sedimentation of part of L. paracasei EV at lower centrifugal forces. This study has solved technically complex problems and essential questions which will facilitate new research focusing on the molecular behavior of probiotics during fermentation and the mechanisms of action mediating the health benefits of fermented products.

Factors Affecting Spontaneous Endocytosis and Survival of Probiotic Lactobacilli in Human Intestinal Epithelial Cells.

Mutualistic bacteria have different forms of interaction with the host. In contrast to the invasion of pathogenic bacteria, naturally occurring internalization of commensal bacteria has not been studied in depth. Three in vitro methods, gentamicin protection, flow cytometry and confocal laser scanning microscopy, have been implemented to accurately assess the internalization of two lactobacillus strains-Lacticaseibacillus paracasei BL23 and Lacticaseibacillus rhamnosus GG-in Caco-2 and T84 intestinal epithelial cells (IECs) under a variety of physiological conditions and with specific inhibitors. First and most interesting, internalization occurred at a variable rate that depends on the bacterial strain and IEC line, and the most efficient was BL23 internalization by T84 and, second, efficient internalization required active IEC proliferation, as it improved naturally at the early confluence stages and by stimulation with epidermal growth factor (EGF). IFN-γ is bound to innate immune responses and autolysis; this cytokine had a significant effect on internalization, as shown by flow cytometry, but increased internalization was not perceived in all conditions, possibly because it was also stimulating autolysis and, as a consequence, the viability of bacteria after uptake could be affected. Bacterial uptake required actin polymerization, as shown by cytochalasin D inhibition, and it was partially bound to clathrin and caveolin dependent endocytosis. It also showed partial inhibition by ML7 indicating the involvement of cholesterol lipid rafts and myosin light chain kinase (MLCK) activation, at least in the LGG uptake by Caco-2. Most interestingly, bacteria remained viable inside the IEC for as long as 72 h without damaging the epithelial cells, and paracellular transcytosis was observed. These results stressed the fact that internalization of commensal and mutualistic bacteria is a natural, nonpathogenic process that may be relevant in crosstalk processes between the intestinal populations and the host, and future studies could determine its connection to processes such as commensal tolerance, resilience of microbial populations or transorganic bacterial migration.

Intake of Natural, Unprocessed Tiger Nuts (Cyperus esculentus L.) Drink Significantly Favors Intestinal Beneficial Bacteria in a Short Period of Time.

Horchata is a natural drink obtained from tiger nut tubers (Cyperus esculentus L.). It has a pleasant milky aspect and nutty flavor; some health benefits have been traditionally attributed to it. This study evaluated the effects of an unprocessed horchata drink on the gut microbiota of healthy adult volunteers (n = 31) who consumed 300 mL of natural, unprocessed horchata with no added sugar daily for 3 days. Although there were no apparent microbial profile changes induced by horchata consumption in the studied population, differences could be determined when volunteers were segmented by microbial clusters. Three distinctive enterogroups were identified previous to consuming horchata, respectively characterized by the relative abundances of Blautia and Lachnospira (B1), Bacteroides (B2) and Ruminococcus and Bifidobacterium (B3). After consuming horchata, samples of all volunteers were grouped into two clusters, one enriched in Akkermansia, Christenellaceae and Clostridiales (A1) and the other with a remarkable presence of Faecalibacterium, Bifidobacterium and Lachnospira (A2). Interestingly, the impact of horchata was dependent on the previous microbiome of each individual, and its effect yielded microbial profiles associated with butyrate production, which are typical of a Mediterranean or vegetable/fiber-rich diet and could be related to the presence of high amylose starch and polyphenols.

Lactobacillus casei extracellular vesicles stimulate EGFR pathway likely due to the presence of proteins P40 and P75 bound to their surface.

In the complex interplay of beneficial bacteria with the host, there are few examples of bacterial metabolites and effector molecules that have been consistently identified. Protective effects on the intestinal epithelium have been ascribed to P40 and P75, two well characterized cell wall muramidases, present in the culture supernatant of strains belonging to the taxon Lactobacillus casei/paracasei/rhamnosus. This work reports that Lactobacillus casei BL23 extracellular vesicles (BL23 EVs) have a small size (17-20 nm or 24-32 nm, depending on the method used) and contain lipoteichoic acid (LTA). Interestingly, all detected P40 and most of P75 were associated to EVs and possibly located at their external surface, as shown by proteinase K digestion. Biosensor assays showed that both proteins bind LTA and vesicles, suggesting that they could bind to ligands like LTA present on BL23 EVs. Native BL23 EVs have a moderate proinflammatory effect and they were able to induce phosphorylation of the epidermal growth factor receptor (EGFR), showing an effect similar to purified P40 and P75 and leading to the conclusion that the activity described in the supernatant (postbiotic) of these bacteria would be mainly due to P40 and P75 bound to EVs.

Intestinal Explant Cultures from Gilthead Seabream (Sparus aurata, L.) Allowed the Determination of Mucosal Sensitivity to Bacterial Pathogens and the Impact of a Plant Protein Diet.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1ß (IL1-ß), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

Differences in the expression of cell envelope proteinases (CEP) in two Lactobacillus paracasei probiotic strains.

Proteinase PrtP (EC:3.4.21.96) is a cell envelope proteinase (CEP) highly expressed in the probiotic strain Lactobacillus paracasei BL312(VSL#3) that accounts for its anti-inflammatory properties. The main aim of this work is to understand differences in CEP expression between this strain and L. paracasei BL23. Hence, differences in the regulation by amino acid sources of four proteinase related genes (prtP, prsA, prtR1 and prtR2) were determined by RT-qPCR in BL312(VSL#3) and BL23 using as a reference BL368, a BL23 derepressed mutant lacking the response regulator (RR) PrcR. BL312(VSL#3) showed greater expression of prtP (2- to 3-fold) than BL23, and prtP was highly repressed by peptone in both strains. Two other putative CEP genes, prtR1 and prtR2, showed a low expression profile. Interestingly, when the prsA-prtP promoter region from both strains, and deleted mutants, were cloned in vector pT1GR, expression of the gfp and mrfp fluorescent reporters was always repressed in BL23 (high or low peptone) and derepressed in BL368, revealing an interesting mechanism of regulation affecting specifically to this promoter. In conclusion, BL312(VSL#3) has higher expression of prtP and other CEP related genes than BL23, that could respond to a natural deregulation in this strain, possibly independent from the RR PrcR.

Ultrasonically-Assisted and Conventional Extraction from Erodium Glaucophyllum Roots Using Ethanol:Water Mixtures: Phenolic Characterization, Antioxidant, and Anti-Inflammatory Activities.

The paper presents experimental results concerning the ultrasonically-assisted extraction of bioactive compounds from Erodium glaucophyllum roots. A comparison with conventional methodology is presented, and thereby the phytochemical composition and the antioxidant and anti-inflammatory activities of extracts are evaluated. The phenolic profile of Erodium extracts was analyzed by TOF-LC-MS-MS. The identification of phenolic compounds revealed that the major component was (+)-gallocatechin in the aqueous extracts obtained for the different extraction methodologies. The highest quantity of phenolic compounds and antioxidant capacity was found in the hydroethanolic extract obtained by conventional extraction (29.22-25.50 mg GAE/g DM; 21.174 mM Trolox equivalent). The highest content of carotenoids, varying from 0.035 to 0.114 mg/g dry matter, was reached by ultrasonic-assisted extraction. Furthermore, Erodium extracts showed a potent inhibition of the inflammatory reaction by means of the inhibition of tumor necrosis factor-alpha (TNF-α). The extracts obtained when ultrasound extraction was combined with ethanol:water (50:50, v/v) presented the greatest inhibition (92%).

Experimental support for multidrug resistance transfer potential in the preterm infant gut microbiota.

BACKGROUND: There is currently a lack of experimental evidence for horizontal gene transfer (HGT) mechanisms in the human gut microbiota. The aim of this study was therefore to experimentally determine the HGT potential in the microbiota of a healthy preterm infant twin pair and to evaluate the global occurrence of the mobilized elements. METHODS: Stool samples were collected. Both shotgun metagenome sequencing and bacterial culturing were done for the same samples. A range of experimental conditions were used to test DNA transfer for the cultured isolates. Searches for global distribution of transferable elements were done for the ~120,000 metagenomic samples in the Sequence Read Archive (SRA) database. RESULTS: DNA transfer experiments demonstrated frequent transmission of an ESBL encoding IncI1 plasmid, a high copy number ColEI plasmid, and bacteriophage P1. Both IncI1 and ColE1 were abundant in the stool samples. In vitro competition experiments showed that transconjugants containing IncI1 plasmids outcompeted the recipient strain in the absence of antibiotic selection. The SRA searches indicated a global distribution of the mobilizable elements, with chicken identified as a possible reservoir for the IncI1 ESBL encoding plasmid. CONCLUSION: Our results experimentally support a major horizontal transmission and persistence potential of the preterm infant gut microbiota mobilome involving genes encoding ESBL.

P40 and P75 Are Singular Functional Muramidases Present in the Lactobacillus casei /paracasei/rhamnosus Taxon.

Lactobacillus casei and Lactobacillus rhamnosus proteins P40 and P75 belong to a large family of secreted cell wall proteins that contain a carboxy(C)-terminal CHAP or NlpC/P60 superfamily domains. In addition to their peptidoglycan hydrolases activity, proteins in this family are specific antigens of pathogens, frequently responsible of interactions with the host. L. rhamnosus GG and L. casei BL23 purified P40 and P75 proteins have antiapoptotic activity by inducing the EGF/Akt pathway. The aim of this work was to study the genetics, phylogeny and dissemination of this family of proteins in the genus Lactobacillus as well as their characteristics and likely function. The scrutiny of their DNA encoding sequences revealed the presence of minisatellite DNA in the P75 encoding gene of L. casei/paracasei strains (cmuB) with intraspecific indels that gave raise to four different alleles (cmuB1-4), which are exclusive of this species. Phylogenic analyses suggest that both proteins are present mainly in the L. casei and Lactobacillus sakei phylogenomic groups. A P40 ancestral gene was possibly present in the common ancestor of Enterococcaceae, Lactobacillaceae and Streptococcaceae. P75 is also present in L. casei and L. sakei groups, but its evolution is difficult to explain only by vertical transmission. Antibodies raised against the N-terminal regions of P40 and P75 improved their immunological detection in culture supernatants as they recognized almost exclusively proteins of L. casei/paracasei/rhamnosus strains, highlighting their structural similarity, that allowed to detect them in different fermented dairy products that contained probiotic L. casei strains. Purified P40 and P75 proteins showed no evident lytic activity but they complemented L. casei BL23 cmuA and cmuB defective mutants, respectively, thus proving that they actively participate in cell division.

Long-term feeding with high plant protein based diets in gilthead seabream (Sparus aurata, L.) leads to changes in the inflammatory and immune related gene expression at intestinal level.

BACKGROUND: In order to ensure sustainability of aquaculture production of carnivourous fish species such as the gilthead seabream (Sparus aurata, L.), the impact of the inclusion of alternative protein sources to fishmeal, including plants, has been assessed. With the aim of evaluating long-term effects of vegetable diets on growth and intestinal status of the on-growing gilthead seabream (initial weight = 129 g), three experimental diets were tested: a strict plant protein-based diet (VM), a fishmeal based diet (FM) and a plant protein-based diet with 15% of marine ingredients (squid and krill meal) alternative to fishmeal (VM+). Intestines were sampled after 154 days. Besides studying growth parameters and survival, the gene expression related to inflammatory response, immune system, epithelia integrity and digestive process was analysed in the foregut and hindgut sections, as well as different histological parameters in the foregut. RESULTS: There were no differences in growth performance (p = 0.2703) and feed utilization (p = 0.1536), although a greater fish mortality was recorded in the VM group (p = 0.0141). In addition, this group reported a lower expression in genes related to pro-inflammatory response, as Interleukine-1ß (il1ß, p = 0.0415), Interleukine-6 (il6, p = 0.0347) and cyclooxigenase-2 (cox2, p = 0.0014), immune-related genes as immunoglobulin M (igm, p = 0.0002) or bacterial defence genes as alkaline phosphatase (alp, p = 0.0069). In contrast, the VM+ group yielded similar survival rate to FM (p = 0.0141) and the gene expression patterns indicated a greater induction of the inflammatory and immune markers (il1ß, cox2 and igm). However, major histological changes in gut were not detected. CONCLUSIONS: Using plants as the unique source of protein on a long term basis, replacing fishmeal in aqua feeds for gilthead seabream, may have been the reason of a decrease in the level of different pro-inflammatory mediators (il1 ß, il6 and cox2) and immune-related molecules (igm and alp), which reflects a possible lack of local immune response at the intestinal mucosa, explaining the higher mortality observed. Krill and squid meal inclusion in vegetable diets, even at low concentrations, provided an improvement in nutrition and survival parameters compared to strictly plant protein based diets as VM, maybe explained by the maintenance of an effective immune response throughout the assay.

The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine.

The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTSLac) EIICB and EIIA domains, respectively, and lacG for the phospho-ß-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTSLac are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG abolishes the growth of L. casei in both disaccharides and analysis of LacG activity showed a high selectivity toward phosphorylated compounds, suggesting that LacG is necessary for the hydrolysis of the intracellular phosphorylated lactose and LacNAc. L. casei (lacAB) strain deficient in galactose-6P isomerase showed a growth rate in lactose (0.0293 ± 0.0014 h-1) and in LacNAc (0.0307 ± 0.0009 h-1) significantly lower than the wild-type (0.1010 ± 0.0006 h-1 and 0.0522 ± 0.0005 h-1, respectively), indicating that their galactose moiety is catabolized through the tagatose-6P pathway. Transcriptional analysis showed induction levels of the lac genes ranged from 130 to 320-fold in LacNAc and from 100 to 200-fold in lactose, compared to cells growing in glucose.

Untargeted metabolomics of fresh and heat treatment Tiger nut (Cyperus esculentus L.) milks reveals further insight into food quality and nutrition.

Tiger nut (Cyperus esculentus L.) is a crop traditionally grown in Valencia Region (Spain) and other temperate and tropical regions in the world, where its tubers are commonly consumed as tiger nut milk (horchata). Because of their nutritive potential and original taste, these products are beginning to spread internationally and, as consequence, analytical procedures to assess nutritional profiles, quality control issues are acquiring increasing relevance. The main objective of this study was to use an advance analytical method and chemometrics tools to determine if the ultra-high temperature (UHT) treatment necessary to extend the shelf life of tiger nut milk would affect the profile of nutrients when compared to fresh product. A cold solvent extraction followed by liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) was used. Datasets obtained from UHT and fresh tiger nut milk data were analyzed through an untargeted metabolomics approach to compare chemical patterns, highlighting differences in citric acid esters of mono- diglycerides (CITREM) and monoacylglycerol (MAG) used as emulsifiers of UHT products, and a remarkably higher abundance of biotin, phosphatidic acid (PA) and L-arginine in fresh products. These results showed that untargeted metabolomics through high resolution tandem mass spectrometry allowed fine differences between food products to be found, therefore, the nutrient lost caused by UHT treatment was clearly discerned.

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

The Potential of Class II Bacteriocins to Modify Gut Microbiota to Improve Host Health.

Production of bacteriocins is a potential probiotic feature of many lactic acid bacteria (LAB) as it can help prevent the growth of pathogens in gut environments. However, knowledge on bacteriocin producers in situ and their function in the gut of healthy animals is still limited. In this study, we investigated five bacteriocin-producing strains of LAB and their isogenic non-producing mutants for probiotic values. The LAB bacteriocins, sakacin A (SakA), pediocin PA-1 (PedPA-1), enterocins P, Q and L50 (enterocins), plantaricins EF and JK (plantaricins) and garvicin ML (GarML), are all class II bacteriocins, but they differ greatly from each other in terms of inhibition spectrum and physicochemical properties. The strains were supplemented to mice through drinking water and changes on the gut microbiota composition were interpreted using 16S rRNA gene analysis. In general, we observed that overall structure of the gut microbiota remained largely unaffected by the treatments. However, at lower taxonomic levels, some transient but advantageous changes were observed. Some potentially problematic bacteria were inhibited (e.g., Staphylococcus by enterocins, Enterococcaceae by GarML, and Clostridium by plantaricins) and the proportion of LAB was increased in the presence of SakA-, plantaricins- and GarML-producing bacteria. Moreover, the treatment with GarML-producing bacteria co-occurred with decreased triglyceride levels in the host mice. Taken together, our results indicate that several of these bacteriocin producers have potential probiotic properties at diverse levels as they promote favorable changes in the host without major disturbance in gut microbiota, which is important for normal gut functioning.

Peptide and amino acid metabolism is controlled by an OmpR-family response regulator in Lactobacillus casei.

A Lactobacillus casei BL23 strain defective in an OmpR-family response regulator encoded by LCABL_18980 (PrcR, RR11), showed enhanced proteolytic activity caused by overexpression of the gene encoding the proteinase PrtP. Transcriptomic analysis revealed that, in addition to prtP expression, PrcR regulates genes encoding peptide and amino acid transporters, intracellular peptidases and amino acid biosynthetic pathways, among others. Binding of PrcR to twelve promoter regions of both upregulated and downregulated genes, including its own promoter, was demonstrated by electrophoretic mobility shift assays showing that PrcR can act as a transcriptional repressor or activator. Phosphorylation of PrcR increased its DNA binding activity and this effect was abolished after replacement of the phosphorylatable residue Asp-52 by alanine. Comparison of the transcript levels in cells grown in the presence or absence of tryptone in the growth medium revealed that PrcR activity responded to the presence of a complex amino acid source in the growth medium. We conclude that the PrcR plays a major role in the control of the peptide and amino acid metabolism in L. casei BL23. Orthologous prcR genes are present in most members of the Lactobacillaceae and Leuconostocaceae families. We hypothesize that they play a similar role in these bacterial groups.

Factors influencing gastrointestinal tract and microbiota immune interaction in preterm infants.

The role of microbial colonization is indispensable for keeping a balanced immune response in life. However, the events that regulate the establishment of the microbiota, their timing, and the way in which they interact with the host are not yet fully understood. Factors such as gestational age, mode of delivery, environment, hygienic measures, and diet influence the establishment of microbiota in the perinatal period. Environmental microbes constitute the most important group of exogenous stimuli in this critical time frame. However, the settlement of a stable gut microbiota in preterm infants is delayed compared to term infants. Preterm infants have an immature gastrointestinal tract and immune system which predisposes to infectious morbidity. Neonatal microbial dynamics and alterations in early gut microbiota may precede and/or predispose to diseases such as necrotizing enterocolitis (NEC), late-onset sepsis or others. During this critical period, nutrition is the principal contributor for immunological and metabolic development, and microbiological programming. Breast milk is a known source of molecules that act synergistically to protect the gut barrier and enhance the maturation of the gut-related immune response. Host-microbe interactions in preterm infants and the protective role of diet focused on breast milk impact are beginning to be unveiled.

Changes in cecal microbiota and mucosal gene expression revealed new aspects of epizootic rabbit enteropathy.

Epizootic Rabbit Enteropathy (ERE) is a severe disease of unknown aetiology that mainly affects post-weaning animals. Its incidence can be prevented by antibiotic treatment suggesting that bacterial elements are crucial for the development of the disease. Microbial dynamics and host responses during the disease were studied. Cecal microbiota was characterized in three rabbit groups (ERE-affected, healthy and healthy pretreated with antibiotics), followed by transcriptional analysis of cytokines and mucins in the cecal mucosa and vermix by q-rtPCR. In healthy animals, cecal microbiota with or without antibiotic pretreatment was very similar and dominated by Alistipes and Ruminococcus. Proportions of both genera decreased in ERE rabbits whereas Bacteroides, Akkermansia and Rikenella increased, as well as Clostridium, γ-Proteobacteria and other opportunistic and pathogenic species. The ERE group displayed remarkable dysbiosis and reduced taxonomic diversity. Transcription rate of mucins and inflammatory cytokines was very high in ERE rabbits, except IL-2, and its analysis revealed the existence of two clearly different gene expression patterns corresponding to Inflammatory and (mucin) Secretory Profiles. Furthermore, these profiles were associated to different bacterial species, suggesting that they may correspond to different stages of the disease. Other data obtained in this work reinforced the notion that ERE morbidity and mortality is possibly caused by an overgrowth of different pathogens in the gut of animals whose immune defence mechanisms seem not to be adequately responding.

Genome-wide expression profiles in very low birth weight infants with neonatal sepsis.

BACKGROUND: Bacterial sepsis is associated with high morbidity and mortality in preterm infants. However, diagnosis of sepsis and identification of the causative agent remains challenging. Our aim was to determine genome-wide expression profiles of very low birth weight (VLBW) infants with and without bacterial sepsis and assess differences. METHODS: This was a prospective observational double-cohort study conducted in VLBW (<1500 g) infants with culture-positive bacterial sepsis and non-septic matched controls. Blood samples were collected as soon as clinical signs of sepsis were identified and before antibiotics were initiated. Total RNA was processed for genome-wide expression analysis using Affymetrix gene arrays. RESULTS: During a 19-month period, 17 septic VLBW infants and 19 matched controls were enrolled. First, a three-dimensional unsupervised principal component analysis based on the entire genome (28 000 transcripts) identified 3 clusters of patients based on gene expression patterns: Gram-positive sepsis, Gram-negative sepsis, and noninfected control infants. Furthermore, these groups were confirmed by using analysis of variance, which identified a transcriptional signature of 554 of genes. These genes had a significantly different expression among the groups. Of the 554 identified genes, 66 belonged to the tumor necrosis factor and 56 to cytokine signaling. The most significantly overexpressed pathways in septic neonates related with innate immune and inflammatory responses and were validated by real-time reverse transcription polymerase chain reaction. CONCLUSIONS: Our preliminary results suggest that genome-wide expression profiles discriminate septic from nonseptic VLBW infants early in the neonatal period. Further studies are needed to confirm these findings.