Aplicación de fluorocromos para el estudio de la viabilidad de las Bacterias Ácido Lácticas (BAL) presentes en productos lácteos

Se estudió la viabilidad de las Bacterias Ácido Lácticas (BAL) procedentes de diversas leches fermentadas de origencomercial, almacenadas en refrigeración a 4ºC. Las bacterias viables y cultivables fueron enumeradas en los mediosTPY (Bifidobacterias), MRS (Lactobacilos) y M17 (Estreptococos). Los recuentos totales (viables y muertas) sellevaron a cabo mediante el uso del sistema comercial LIVE/DEAD® Bac LightTM VIABILITY. La viabilidad de lasBAL fue estudiada antes y después de la fecha de caducidad de los productos lácteos. Las bacterias procedentes delos distintos productos analizados presentaron diferencias en su viabilidad. Las BAL deben permanecer vivas en losproductos para poder ejercer beneficios para la salud tras el consumo de los mismos. La técnica de recuentomicrobiano basada en el uso de fluorocromos, es una vía rápida y efectiva para la enumeración de microorganismosviables y no viables en productos lácteos, evitando la utilización de los tediosos medios tradicionales

HPV infection and bacterial microbiota in breast milk and infant oral mucosa.

OBJECTIVE: We investigated the association between bacterial microbiota in breast milk and the infant mouth. The influence of human papilloma virus (HPV) infection on infant oral microbiota was also assessed.MATERIAL AND METHODS: Altogether 35 breast milk and 35 infant oral samples with known HPV status were selected from the Finnish Family HPV Study cohort. In total, there were 31 mother-infant pairs. The microbiota composition was characterized by 16S rRNA gene sequencing (V3-V4 region).RESULTS: HPV DNA was present in 8.6% (3/35) of the breast milk and 40% (14/35) of the infant oral samples. Eight shared genera between breast milk and infant oral were found; these included Streptococcus, Staphylococcus, Unclassified Gemellaceae, Rothia, Veillonella, Haemophilus, Propionibacterium and Corynebacterium. HPV status was not associated with either microbiota richness or diversity in the infant mouth. However, the infant oral microbiota clustered in different groups according to HPV status. We detected higher abundance of Veillonella dispar (p = 0.048) at species level in HPV negative infant oral samples. We did not detect differences in the breast milk microbiota composition related to HPV infection due to only three HPV positive milk samples.CONCLUSIONS: HPV infection is associated with distinct oral bacterial microbiota composition in infants. The direction of causality underlying the phenomenon remains unclear.</p

Complex Polysaccharide-Based Nanocomposites for Oral Insulin Delivery

Polyelectrolyte nanocomposites rarely reach a stable state and aggregation often occurs. Here, we report the synthesis of nanocomposites for the oral delivery of insulin composed of alginate, dextran sulfate, poly-(ethylene glycol) 4000, poloxamer 188, chitosan, and bovine serum albumin. The nanocomposites were obtained by Ca2+-induced gelation of alginate followed by an electrostatic-interaction process among the polyelectrolytes. Chitosan seemed to be essential for the final size of the nanocomposites and there was an optimal content that led to the synthesis of nanocomposites of 400–600 nm hydrodynamic size. The enhanced stability of the synthesized nanocomposites was assessed with LUMiSizer after synthesis. Nanocomposite stability over time and under variations of ionic strength and pH were assessed with dynamic light scattering. The rounded shapes of nanocomposites were confirmed by scanning electron microscopy. After loading with insulin, analysis by HPLC revealed complete drug release under physiologically simulated conditions

Bifidobacterium longum CECT 7347 Modulates Immune Responses in a Gliadin-Induced Enteropathy Animal Model

Coeliac disease (CD) is an autoimmune disorder triggered by gluten proteins (gliadin) that involves innate and adaptive immunity. In this study, we hypothesise that the administration of Bifidobacterium longum CECT 7347, previously selected for reducing gliadin immunotoxic effects in vitro, could exert protective effects in an animal model of gliadin-induced enteropathy. The effects of this bacterium were evaluated in newborn rats fed gliadin alone or sensitised with interferon (IFN)-γ and fed gliadin. Jejunal tissue sections were collected for histological, NFκB mRNA expression and cytokine production analyses. Leukocyte populations and T-cell subsets were analysed in peripheral blood samples. The possible translocation of the bacterium to different organs was determined by plate counting and the composition of the colonic microbiota was quantified by real-time PCR. Feeding gliadin alone reduced enterocyte height and peripheral CD4+ cells, but increased CD4+/Foxp3+ T and CD8+ cells, while the simultaneous administration of B. longum CECT 7347 exerted opposite effects. Animals sensitised with IFN-γ and fed gliadin showed high cellular infiltration, reduced villi width and enterocyte height. Sensitised animals also exhibited increased NFκB mRNA expression and TNF-α production in tissue sections. B. longum CECT 7347 administration increased NFκB expression and IL-10, but reduced TNF-α, production in the enteropathy model. In sensitised gliadin-fed animals, CD4+, CD4+/Foxp3+ and CD8+ T cells increased, whereas the administration of B. longum CECT 7347 reduced CD4+ and CD4+/Foxp3+ cell populations and increased CD8+ T cell populations. The bifidobacterial strain administered represented between 75–95% of the total bifidobacteria isolated from all treated groups, and translocation to organs was not detected. These findings indicate that B. longum attenuates the production of inflammatory cytokines and the CD4+ T-cell mediated immune response in an animal model of gliadin-induced enteropathy

Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

Development of the preterm gut microbiome in twins at risk of necrotising enterocolitis and sepsis

The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples (n = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM (n = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae. The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance of Escherichia sp. preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducing Escherichia sp. and increasing other Enterobacteriaceae. This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics

Evaluating amplified rDNA restriction analysis assay for identification of bacterial communities

Amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism were originally used for strain typing and for screening clone libraries to identify phylogenetic clusters within a microbial community. Here we used ARDRA as a model to examine the capacity of restriction-based techniques for clone identification, and the possibility of deriving phylogenetic information from ARDRA-based dendrograms. ARDRA was performed in silico on 48,759 sequences from the Ribosomal Database Project, and it was found that the fragmentation profiles were not necessarily unique for each sequence in the database, resulting in different species sharing fragmentation profiles. Although ARDRA-based clusters separated clones into different genera, these phylogenetic clusters did not overlap with trees constructed according to sequence alignment, calling into question the intra-genus ARDRA-based phylogeny. It is thus suggested that the prediction power of ARDRA clusters in identifying clone phylogeny be regarded with caution

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.M.C. acknowledges a “Rio Hortega” Research Fellowship Grant (CM13/0017) and M.V. acknowledges grants PI11/0313 and RD12/0026/0012 (Red SAMID) from the Instituto Carlos III (Spanish Ministry of Economy and Competitivity). M.C.C. and G.P-M. were supported by the grant AGL2013-47420-R from the Spanish Ministry of Science and Innovation.Peer reviewe

Mycobiome Profiles in Breast Milk from Healthy Women Depend on Mode of Delivery, Geographic Location, and Interaction with Bacteria

Recent studies report the presence of fungal species in breast milk of healthy mothers, suggesting a potential role in infant mycobiome development. In the present work, we aimed to determine whether the healthy human breast milk mycobiota is influenced by geographical location and mode of delivery, as well as to investigate its interaction with bacterial profiles in the same samples. A total of 80 mature breast milk samples from 4 different countries were analyzed by Illumina sequencing of the internal transcribed spacer 1 (ITS1) region, joining the 18S and 5.8S regions of the fungal rRNA region. Basidiomycota and Ascomycota were found to be the dominant phyla, with Malassezia and Davidiella being the most prevalent genera across countries. A core formed by Malassezia, Davidiella, Sistotrema, and Penicillium was shared in the milk samples from the different origins, although specific shifts in mycobiome composition were associated with geographic location and delivery mode. The presence of fungi in the breast milk samples was further confirmed by culture and isolate characterization, and fungal loads were estimated by quantitative PCR (qPCR) targeting the fungal ITS1 region. Cooccurrence network analysis of bacteria and fungi showed complex interactions that were influenced by geographical location, mode of delivery, maternal age, and pregestational body mass index. The presence of a breast milk mycobiome was confirmed in all samples analyzed, regardless of the geographic origin.IMPORTANCE During recent years, human breast milk has been documented as a potential source of bacteria for the newborn. Recently, we have reported the presence of fungi in breast milk from healthy mothers. It is well known that environmental and perinatal factors can affect milk bacteria; however, the impact on milk fungi is still unknown. The current report describes fungal communities (mycobiota) in breast milk samples across different geographic locations and the influence of the mode of delivery. We also provide novel insights on bacterium-fungus interactions, taking into account environmental and perinatal factors. We identified a core of four genera shared across locations, consisting of Malassezia, Davidiella, Sistotrema, and Penicillium, which have been reported to be present in the infant gut. Our data confirm the presence of fungi in breast milk across continents and support the potential role of breast milk in the initial seeding of fungal species in the infant gut