Early-Life Development of the Bifidobacterial Community in the Infant Gut.

The establishment of the gut microbiota poses implications for short and long-term health. Bifidobacterium is an important taxon in early life, being one of the most abundant genera in the infant intestinal microbiota and carrying out key functions for maintaining host-homeostasis. Recent metagenomic studies have shown that different factors, such as gestational age, delivery mode, or feeding habits, affect the gut microbiota establishment at high phylogenetic levels. However, their impact on the specific bifidobacterial populations is not yet well understood. Here we studied the impact of these factors on the different Bifidobacterium species and subspecies at both the quantitative and qualitative levels. Fecal samples were taken from 85 neonates at 2, 10, 30, 90 days of life, and the relative proportions of the different bifidobacterial populations were assessed by 16S rRNA-23S rRNA internal transcribed spacer (ITS) region sequencing. Absolute levels of the main species were determined by q-PCR. Our results showed that the bifidobacterial population establishment is affected by gestational age, delivery mode, and infant feeding, as it is evidenced by qualitative and quantitative changes. These data underline the need for understanding the impact of perinatal factors on the gut microbiota also at low taxonomic levels, especially in the case of relevant microbial populations such as Bifidobacterium. The data obtained provide indications for the selection of the species best suited for the development of bifidobacteria-based products for different groups of neonates and will help to develop rational strategies for favoring a healthy early microbiota development when this process is challenged.

Vitamin D deficiency in children: a challenging diagnosis!

The concern about the assessment of vitamin D status is growing. Numerous publications warn about the high prevalence of vitamin D deficiency, as well as the potential role of vitamin D in non-bone health outcomes. The status of vitamin D is usually assessed by measuring serum total 25-hydroxyvitamin D (25OHD) concentration. This is the major circulating form of vitamin D and keeps an inverse correlation with serum parathyroid hormone (PTH) concentration. A value of 25OHD of 20 ng/ml is generally assumed as threshold of vitamin D sufficiency in epidemiologic studies because serum PTH tends to increase when the 25OHD concentration stands below this value. In pediatric population, very few studies have analyzed this issue and the negative relationship between serum 25OHD and serum PTH is not clear, which is the suitable circulating concentration of 25OHD and the threshold of deficiency being matters of controversy. The majority of 25OHD circulates in serum tightly bound to a globulin (DBP). According to the free hormone hypothesis, protein-bound hormones are not biologically available and it is the free form that exerts or facilitates the physiologic actions. If this is true, factors that affect DBP may alter the interpretation of total serum 25OHD measurements.

Hypophosphatemia and growth.

Over the last decade the discovery of fibroblast growth factor 23 (FGF23) and the progressive and ongoing clarification of its role in phosphate and mineral metabolism have led to expansion of the diagnostic spectrum of primary hypophosphatemic syndromes. This article focuses on the impairment of growth in these syndromes. Growth retardation is a common, but not constant, feature and it presents with large variability. As a result of the very low prevalence of other forms of primary hypophosphatemic syndromes, the description of longitudinal growth and the pathogenesis of its impairment have been mostly studied in X-linked hypophosphatemia (XLH) patients and in Hyp mice, the animal model of this disease. In general, children with XLH have short stature with greater shortness of lower limbs than trunk. Treatment with phosphate supplements and 1α vitamin D derivatives heals active lesions of rickets, but does not normalize growth of XLH patients. Patients might benefit from recombinant human growth hormone (rhGH) therapy, which may accelerate the growth rate without increasing body disproportion or correcting hypophosphatemia. These clinical data as well as research findings obtained in Hyp mice suggest that the pathogenesis of defective growth in XLH and other hypophosphatemic syndromes is not entirely dependent on the mineralization disorder and point to other effects of hypophosphatemia itself or FGF23 on the metabolism of bone and growth plate.

Old Folks, Bad Boon: Antimicrobial Resistance in the Infant Gut Microbiome.

The development of the intestinal microbiome in the neonate starts, mainly, at birth, when the infant receives its founding microbial inoculum from the mother. This microbiome contains genes conferring resistance to antibiotics since these are found in some of the microorganisms present in the intestine. Similarly to microbiota composition, the possession of antibiotic resistance genes is affected by different perinatal factors. Moreover, antibiotics are the most used drugs in early life, and the use of antibiotics in pediatrics covers a wide variety of possibilities and treatment options. The disruption in the early microbiota caused by antibiotics may be of great relevance, not just because it may limit colonization by beneficial microorganisms and increase that of potential pathogens, but also because it may increase the levels of antibiotic resistance genes. The increase in antibiotic-resistant microorganisms is one of the major public health threats that humanity has to face and, therefore, understanding the factors that determine the development of the resistome in early life is of relevance. Recent advancements in sequencing technologies have enabled the study of the microbiota and the resistome at unprecedent levels. These aspects are discussed in this review as well as some potential interventions aimed at reducing the possession of resistance genes.

In Vitro Probiotic Modulation of the Intestinal Microbiota and 2'Fucosyllactose Consumption in Fecal Cultures from Infants at Two Months of Age.

2'-fucosyllactose (2'FL) is one of the most abundant oligosaccharides in human milk, with benefits on neonatal health. Previous results point to the inability of the fecal microbiota from some infants to ferment 2'FL. We evaluated a probiotic formulation, including the strains Lactobacillus helveticus Rosell®-52 (R0052), Bifidobacterium longum subsp. infantis Rosell®-33 (R0033), and Bifidobacterium bifidum Rosell®-71 (R0071), individually or in an 80:10:10 combination on the microbiota and 2'FL degradation. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed) with added probiotic formulation or the constituent strains in the presence of 2'FL. Microbiota composition was analyzed by 16S rRNA gene sequencing. Gas accumulation, pH decrease and 2'FL consumption, and levels of different metabolites were determined by chromatography. B. bifidum R0071 was the sole microorganism promoting a partial increase of 2'FL degradation during fermentation in fecal cultures of 2'FL slow-degrading donors. However, major changes in microbiota composition and metabolic activity occurred with L. helveticus R0052 or the probiotic formulation in cultures of slow degraders. Further studies are needed to decipher the role of the host intestinal microbiota in the efficacy of these strains.

Effect of Intrapartum Antibiotics Prophylaxis on the Bifidobacterial Establishment within the Neonatal Gut.

Antibiotics are important disruptors of the intestinal microbiota establishment, linked to immune and metabolic alterations. The intrapartum antibiotics prophylaxis (IAP) is a common clinical practice that is present in more than 30% of labours, and is known to negatively affect the gut microbiota composition. However, little is known about how it affects to Bifidobacterium (sub)species level, which is one of the most important intestinal microbial genera early in life. This study presents qualitative and quantitative analyses of the bifidobacterial (sub)species populations in faecal samples, collected at 2, 10, 30 and 90 days of life, from 43 healthy full-term babies, sixteen of them delivered after IAP use. This study uses both 16S rRNA-23S rRNA internal transcribed spacer (ITS) region sequencing and q-PCR techniques for the analyses of the relative proportions and absolute levels, respectively, of the bifidobacterial populations. Our results show that the bifidobacterial populations establishment is affected by the IAP at both quantitative and qualitative levels. This practice can promote higher bifidobacterial diversity and several changes at a compositional level. This study underlines specific targets for developing gut microbiota-based products for favouring a proper bifidobacterial microbiota development when IAP is required.

Influence of 2'-Fucosyllactose on the Microbiota Composition and Metabolic Activity of Fecal Cultures from Breastfed and Formula-Fed Infants at Two Months of Age.

Although breast milk is considered the gold standard of nutrition for infant feeding, some circumstances may make breastfeeding difficult. Several commercial milk preparations include synthetic human milk oligosaccharides (HMOs) in their composition. However, the effect of HMOs on the establishment of the intestinal microbiota remains incompletely understood. Independent batch fermentations were performed with feces from six full-term infant donors of two months of age (three breastfed and three formula-fed, exclusively) in the presence of 2'fucosyllactose (2'FL), one of the most abundant HMOs in human milk. Microbiota composition was analyzed by 16S rRNA gene sequencing at baseline and at 24 h of incubation. The 2'FL consumption, gas accumulation, and levels of different metabolites were determined by chromatography. Microbiota profiles at baseline were clearly influenced by the mode of feeding and by the intrinsic ability of microbiotas to degrade 2'FL. The 2'FL degradation rate clustered fecal cultures into slow and fast degraders, regardless of feeding type, this being a determinant factor influencing the evolution of the microbiota during incubation, although the low number of donors precludes drawing sound conclusions. More studies are needed to decipher the extent to which the early intervention with HMOs could influence the microbiota as a function of its ability to utilize 2'FL.

Phenotyping and relative quantification of vitamin D binding protein in a paediatric population by using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Adequate concentrations of vitamin D are required to ensure bone health and minimize the incidence of multiple extraskeletal diseases. Although total 25-hydroxyvitamin D (25OHD) remains the recommended biomarker for assessing vitamin D status, it has been speculated that free 25OHD correlates better with clinical outcomes. The calculation of free 25OHD depends on the concentrations of vitamin D binding protein (DBP), the determination of which involves different immunoassays and has led to varying results and conclusions. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and relative quantification of DBP isoforms. METHODS: We used serum samples from healthy children ( n = 79), mainly Caucasian (88%). Proteins were denatured, reduced, alkylated and digested with trypsin. Purified peptides were analysed by LC-MS/MS. The DBP phenotype was established by using the combinations of tryptic peptides associated with each of the three isoforms and one peptide common to all of them to perform relative quantification. The genotyping of volunteers ( n = 7) facilitated verification of the ability of our method to correctly identify the DBP phenotype. RESULTS: The DBP phenotype was correctly established in all samples from volunteers, based on the 100% correlation observed with the genotype. The most common DBP phenotype in Caucasian children was 2/1S (34%) and the rarest 1F/1F (2%). The relative quantification of DBP concentrations did not show statistically significant differences between phenotypes ( P = 0.11). CONCLUSIONS: LC-MS/MS enabled simultaneous phenotyping and relative quantification of DBP, while avoiding the analytical limitations of immunoassays and confirming similar concentrations of DBP in all phenotypes.

Effect of sludge features and extraction-esterification technology on the synthesis of biodiesel from secondary wastewater treatment sludges.

Secondary sludge from municipal wastewater treatment plant is proposed as a promising alternative lipid feedstock for biodiesel production. A deep study combining different type of raw materials (sludge coming from the oxic, anoxic and anaerobic steps of the biological treatment) with different technologies (liquid-liquid and solid-liquid extractions followed by acid catalysed transesterification and in situ extraction-transesterification procedure) allows a complete comparison of available technologies. Different parameters - contact time, catalyst concentration, pretreatments - were considered, obtaining more than 17% FAMEs yield after 50min of sonication with the in situ procedure and 5% of H2SO4. This result corresponds to an increment of more than 65% respect to the best results reported at typical conditions. Experimental data were used to propose a mathematical model for this process, demonstrating that the mass transfer of lipids from the sludge to the liquid is the limiting step.

Marker of vitamin D status in healthy children: Free or total 25-hydroxyvitamin D?

OBJECTIVE: To assess if serum free 25-hydroxyvitamin D (25OHD) is a better indicator of vitamin D status than total 25OHD in healthy children. METHODS: Cross-sectional prospective clinical study was designed. We measured serum free 25OHD concentrations and its correlation with calculated free 25OHD, total 25OHD, intact parathyroid hormone (PTH), and vitamin D binding protein (DBP) in children. The influence of age, sex, ethnicity, body mass index (BMI), season of the year, diet intake, vitamin supplements, time spent outdoors and albumin concentrations on free 25OHD was also analyzed. 241 children aged from 0 days to 14 years, and living in the northern Spain (latitude 43° N), were included. RESULTS: Mean (SD) free 25OHD concentrations were 2.48 (1.39), 5.46 (3.12), 4.12 (1,72), 3.82 (1.43) pg/ml in children aged 0 days, 1 month-2 years, 2-6 years and >6 years, respectively. Correlation between directly measured and calculated free 25OHD was high and significant (r = 0.66) as well as the correlation between serum free and total 25OHD concentrations (r = 0.61). No significant correlation was found between PTH and free 25OHD (r = -0.08). The total 25OHD and PTH concentrations' correlation was inverse (r = -0.25) and significant. Neither free nor total 25OHD concentrations correlated with DBP concentrations. Among the analyzed variables, free 25OHD values were higher in spring/summer than in autumn/winter in children older than 6 years. CONCLUSIONS: : These findings do not support that free 25OHD is a better marker of vitamin D deficiency than total 25OHD in healthy pediatric population.