Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

Immune Responsiveness to LPS Determines Risk of Childhood Wheeze and Asthma in 17q21 Risk Allele Carriers.

Rationale: In murine models, microbial exposures induce protection from experimental allergic asthma through innate immunity. Objectives: Our aim was to assess the association of early life innate immunity with the development of asthma in children at risk. Methods: In the PASTURE farm birth cohort, innate T-helper cell type 2 (Th2), Th1, and Th17 cytokine expression at age 1 year was measured after stimulation of peripheral blood mononuclear cells with LPS in n = 445 children. Children at risk of asthma were defined based on single-nucleotide polymorphisms at the 17q21 asthma gene locus. Specifically, we used the SNP rs7216389 in the GSDMB gene. Wheeze in the first year of life was assessed by weekly diaries and asthma by questionnaire at age 6 years. Measurements and Main Results: Not all cytokines were detectable in all children after LPS stimulation. When classifying detectability of cytokines by latent class analysis, carrying the 17q21 risk allele rs7216389 was associated with risk of wheeze only in the class with the lowest level of LPS-induced activation: odds ratio (OR), 1.89; 95% confidence interval [CI], 1.13-3.16; P = 0.015. In contrast, in children with high cytokine activation after LPS stimulation, no association of the 17q21 risk allele with wheeze (OR, 0.63; 95% CI, 0.29-1.40; P = 0.258, P = 0.034 for interaction) or school-age asthma was observed. In these children, consumption of unprocessed cow's milk was associated with higher cytokine activation (OR, 3.37; 95% CI, 1.56-7.30; P = 0.002), which was in part mediated by the gut microbiome. Conclusions: These findings suggest that within the 17q21 genotype, asthma risk can be mitigated by activated immune responses after innate stimulation, which is partly mediated by a gut-immune axis.

The developing gut-lung axis: postnatal growth restriction, intestinal dysbiosis, and pulmonary hypertension in a rodent model.

BACKGROUND: Postnatal growth restriction (PNGR) in premature infants increases risk of pulmonary hypertension (PH). In a rodent model, PNGR causes PH, while combining PNGR and hyperoxia increases PH severity. We hypothesized that PNGR causes intestinal dysbiosis and that treatment with a probiotic attenuates PNGR-associated PH. METHOD: Pups were randomized at birth to room air or 75% oxygen (hyperoxia), to normal milk intake (10 pups/dam) or PNGR (17 pups/dam), and to probiotic Lactobacillus reuteri DSM 17938 or phosphate-buffered saline. After 14 days, PH was assessed by echocardiography and right ventricular hypertrophy (RVH) was assessed by Fulton's index (right ventricular weight/left ventricle + septal weight). The small bowel and cecum were analyzed by high-throughput 16S ribosomal RNA gene sequencing. RESULTS: PNGR with or without hyperoxia (but not hyperoxia alone) altered the microbiota of the distal small bowel and cecum. Treatment with DSM 17938 attenuated PH and RVH in pups with PNGR, but not hyperoxia alone. DSM 17938 treatment decreased α-diversity. The intestinal microbiota differed based on oxygen exposure, litter size, and probiotic treatment. CONCLUSION: PNGR causes intestinal dysbiosis and PH. Treatment with DSM 17938 prevents PNGR-associated RVH and PH. Changes in the developing intestine and intestinal microbiota impact the developing lung vasculature and RV.

Neonatal Vitamin A Supplementation and Vitamin A Status Are Associated with Gut Microbiome Composition in Bangladeshi Infants in Early Infancy and at 2 Years of Age.

BACKGROUND: Infancy is a crucial period for establishing the intestinal microbiome. This process may be influenced by vitamin A (VA) status because VA affects intestinal immunity and epithelial integrity, factors that can, in turn, modulate microbiome development. OBJECTIVES: The aim of this study was to determine if neonatal VA supplementation (VAS) affected the abundance of Bifidobacterium, a beneficial commensal, or of Proteobacteria, a phylum containing enteric pathogens, in early (6-15 wk) or late (2 y) infancy. Secondary objectives were to determine if VAS affected the abundance of other bacterial taxa, and to determine if VA status assessed by measuring plasma retinol was associated with bacterial abundance. METHODS: Three hundred and six Bangladeshi infants were randomized by sex and birthweight status (above/below median) to receive 1 VA dose (50,000 IU) or placebo within 48 h of birth. Relative abundance at the genus level and above was assessed by 16S rRNA gene sequencing. A terminal restriction fragment-length polymorphism assay was used to identify Bifidobacterium species and subspecies at 6 wk. RESULTS: Linear regression showed that Bifidobacterium abundance in early infancy was lower in boys (median, 1st/3rd quartiles; 0.67, 0.52/0.78) than girls (0.73, 0.60/0.80; P = 0.003) but that boys receiving VAS (0.69, 0.55/0.78) had higher abundance than boys receiving placebo (0.65, 0.44/0.77; P = 0.039). However this difference was not seen in girls (VAS 0.71, 0.54/0.80; placebo 0.75, 0.63/0.81; P = 0.25). VAS did not affect Proteobacteria abundance. Sex-specific associations were also seen for VA status, including positive associations of plasma retinol with Actinobacteria (the phylum containing Bifidobacterium) and Akkermansia, another commensal with possible health benefits, for girls in late infancy. CONCLUSIONS: Better VA status in infancy may influence health both in infancy and later in life by promoting the establishment of a healthy microbiota. This postulated effect of VA may differ between boys and girls. This trial was registered at clinicaltrials.gov as NCT02027610.

Western Diet-Induced Dysbiosis in Farnesoid X Receptor Knockout Mice Causes Persistent Hepatic Inflammation after Antibiotic Treatment.

Patients who have liver cirrhosis and liver cancer also have reduced farnesoid X receptor (FXR). The current study analyzes the effect of diet through microbiota that affect hepatic inflammation in FXR knockout (KO) mice. Wild-type and FXR KO mice were on a control (CD) or Western diet (WD) for 10 months. In addition, both CD- and WD-fed FXR KO male mice, which had hepatic lymphocyte and neutrophil infiltration, were treated by vancomycin, polymyxin B, and Abx (ampicillin, neomycin, metronidazole, and vancomycin). Mice were subjected to morphological analysis as well as gut microbiota and bile acid profiling. Male WD-fed FXR KO mice had the most severe steatohepatitis. FXR KO also had reduced Firmicutes and increased Proteobacteria, which could be reversed by Abx. In addition, Abx eliminated hepatic neutrophils and lymphocytes in CD-fed, but not WD-fed, FXR KO mice. Proteobacteria and Bacteroidetes persisted in WD-fed FXR KO mice even after Abx treatment. Only polymyxin B could reduce hepatic lymphocytes in WD-fed FXR KO mice. The reduced hepatic inflammation by antibiotics was accompanied by decreased free and conjugated secondary bile acids as well as changes in gut microbiota. Our data revealed that Lactococcus, Lactobacillus, and Coprococcus protect the liver from inflammation.

Hepatic inflammation caused by dysregulated bile acid synthesis is reversible by butyrate supplementation.

Dysregulated bile acid (BA) synthesis or reduced farnesoid X receptor (FXR) levels are found in patients having metabolic diseases, autoimmune hepatitis, and liver cirrhosis or cancer. The objective of this study was to establish the relationship between butyrate and dysregulated BA synthesis-induced hepatitis as well as the effect of butyrate in reversing the liver pathology. Wild-type (WT) and FXR knockout (KO) male mice were placed on a control (CD) or western diet (WD) for 15 months. In the presence or absence of butyrate supplementation, feces obtained from 15-month-old WD-fed FXR KO mice, which had severe hepatitis and liver tumors, were transplanted to 7-month-old WD-fed FXR KO for 3 months. Hepatic phenotypes, microbiota profile, and BA composition were analyzed. Butyrate-generating bacteria and colonic butyrate concentration were reduced due to FXR inactivation and further reduced by WD intake. In addition, WD-fed FXR KO male mice had the highest concentration of hepatic ß-muricholic acid (ß-MCA) and bacteria-generated deoxycholic acid (DCA) accompanied by serious hepatitis. Moreover, dysregulated BA and reduced SCFA signaling co-existed in both human liver cancers and WD-fed FXR KO mice. Microbiota transplantation using butyrate-deficient feces derived from 15-month-old WD-fed FXR KO mice increased hepatic lymphocyte numbers as well as hepatic ß-MCA and DCA concentrations. Furthermore, butyrate supplementation reduced hepatic ß-MCA as well as DCA and eliminated hepatic lymphocyte infiltration. In conclusion, reduced butyrate contributes to the development of hepatitis in the FXR KO mouse model. In addition, butyrate reverses dysregulated BA synthesis and its associated hepatitis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Digestion of Human Milk Oligosaccharides by Bifidobacterium breve in the Premature Infant.

OBJECTIVE: The aim of this study was to measure consumption and absorption of human milk oligosaccharides (HMOs) in a cohort of premature infants treated with probiotic Bifidobacterium breve. METHODS: Twenty-nine premature infants (median gestational age 28 weeks, range 23-32 weeks) cared for in the neonatal intensive care unit of the King Edward and Princess Margaret Hospital in Perth, Australia, were treated with B breve at a dose of 1.66 billion organisms per day. Samples of feces, urine, and milk were obtained at initiation of the probiotic and again 3 weeks later. 16S ribosomal RNA from the feces was analyzed by next-generation sequencing. Quantitation of HMO content of the milk, urine, and feces was performed using nano-high-performance liquid chromatography-chip/time-of-flight mass spectrometry. RESULTS: There was heterogeneity in colonization with bifidobacteria. "Responders" received milk with higher percentages of fucosylated HMOs and had higher percentages of bifidobacteria and lower percentages of Enterobacteriaceae in their feces than "nonresponders." Several individual HMOs in the milk were associated with changes in fecal bifidobacteria over time. Changes over time in milk, fecal, and urine HMOs suggested heterogeneity among HMO structures in consumption by microbes in the gut lumen and absorption from the intestine. CONCLUSIONS: Colonization of the premature infant intestinal tract with probiotic B breve is influenced by prebiotic HMOs. B breve is a selective consumer of HMOs in the premature infant.

Bovine milk oligosaccharides decrease gut permeability and improve inflammation and microbial dysbiosis in diet-induced obese mice.

Obesity is characterized by altered gut homeostasis, including dysbiosis and increased gut permeability closely linked to the development of metabolic disorders. Milk oligosaccharides are complex sugars that selectively enhance the growth of specific beneficial bacteria in the gastrointestinal tract and could be used as prebiotics. The aim of the study was to demonstrate the effects of bovine milk oligosaccharides (BMO) and Bifidobacterium longum ssp. infantis (B. infantis) on restoring diet-induced obesity intestinal microbiota and barrier function defects in mice. Male C57/BL6 mice were fed a Western diet (WD, 40% fat/kcal) or normal chow (C, 14% fat/kcal) for 7 wk. During the final 2 wk of the study, the diet of a subgroup of WD-fed mice was supplemented with BMO (7% wt/wt). Weekly gavage of B. infantis was performed in all mice starting at wk 3, yet B. infantis could not be detected in any luminal contents when mice were killed. Supplementation of the WD with BMO normalized the cecal and colonic microbiota with increased abundance of Lactobacillus compared with both WD and C mice and restoration of Allobaculum and Ruminococcus levels to that of C mice. The BMO supplementation reduced WD-induced increase in paracellular and transcellular flux in the large intestine as well as mRNA levels of the inflammatory marker tumor necrosis factor α. In conclusion, BMO are promising prebiotics to modulate gut microbiota and intestinal barrier function for enhanced health.

Probiotic Administration in Infants With Gastroschisis: A Pilot Randomized Placebo-Controlled Trial.

OBJECTIVES: Infants with gastroschisis often require long periods of gastric suctioning and hospitalization. The impact of these interventions on the intestinal microbiota and attempts to alter the microbial community have not been studied. We sought to determine how the intestinal microbiota is influenced by the current treatment of gastroschisis and whether alteration of the intestinal microbiota with a probiotic microbe will influence length of hospitalization. METHODS: We performed a randomized, placebo-controlled pilot study of administration of probiotic Bifidobacterium longum subsp. infantis in 24 infants with gastroschisis. The primary outcome was changes in the fecal microbiota, and the secondary outcome was length of hospital stay. RESULTS: Administration of the probiotic or placebo was well tolerated, even during the period of gastric suctioning. The overall microbial communities were not significantly different between groups, although analysis of the final specimens by family demonstrated higher Bifidobacteriaceae, lower Clostridiaceae, and trends toward lower Enterobacteriaceae, Enterococcaceae, Staphylococcaceae, and Streptococcaceae in the probiotic group. Clinical outcomes, including length of hospital stay, did not differ between groups. CONCLUSIONS: In this pilot study, there was significant in infants with gastroschisis that was partially attenuated by the administration of B longum subsp. infantis.

Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota: a proof-of-concept study.

Human milk oligosaccharides (HMOs) play a key role in shaping and maintaining a healthy infant gut microbiota. This article demonstrates the potential of combining recent advances in glycomics and genomics to correlate abundances of fecal microbes and fecal HMOs. Serial fecal specimens from two healthy breast-fed infants were analyzed by bacterial DNA sequencing to characterize the microbiota and by mass spectrometry to determine abundances of specific HMOs that passed through the intestinal tract without being consumed by the luminal bacteria. In both infants, the fecal bacterial population shifted from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life. An initial rise in fecal HMOs corresponded with bacterial populations composed primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation revealed that HMO consumption is highly structure-specific, with unique isomers being consumed and others passing through the gut unaltered. These results represent a proof-of-concept and are consistent with the highly selective, prebiotic effect of HMOs in shaping the gut microbiota in the first weeks of life. The analysis of selective fecal bacterial substrates as a measure of alterations in the gut microbiota may be a potential marker of dysbiosis.

Human milk oligosaccharides in premature infants: absorption, excretion, and influence on the intestinal microbiota.

BACKGROUND: Human milk oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis (NEC) and sepsis, and the influence of HMOs on the microbiota is unclear. METHODS: Milk, urine, and stool specimens from 14 mother-premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing to complement a previous analysis. RESULTS: Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces, and milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes were noted in premature infants of nonsecretor mothers. Specific HMO structures in the milk, urine, and feces were associated with alterations in fecal Proteobacteria and Firmicutes. CONCLUSION: HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and NEC, while other HMOs may increase dysbiosis in this population.

Analysis of raw goat milk microbiota: impact of stage of lactation and lysozyme on microbial diversity.

To protect infants from infection, human milk contains high levels of the enzyme lysozyme, unlike the milk of dairy animals. We have genetically engineered goats to express human lysozyme (hLZ milk) in their milk at 68% the amount found in human milk to help extend this protection. This study looked at the effect of hLZ on bacteria in raw milk over time. As the microbial diversity of goats' milk has yet to be investigated in depth using next-generation sequencing (NGS) technologies, we applied NGS and clone library sequencing (CLS) to determine the microbiota of raw goat milk (WT milk) and hLZ milk at early, mid and late lactation. Overall, in WT milk, the bacterial populations in milk at early and mid lactation were similar to each other with a shift occurring at late lactation. Both methods found Proteobacteria as the dominant bacteria at early and mid lactation, while Actinobacteria surged at late lactation. These changes were related to decreases in Pseudomonas and increases in Micrococcus. The bacterial populations in hLZ milk were similar to WT milk at early and mid lactation with the only significant differences occurring at late lactation with the elevation of Bacillaceae, Alicyclobacillaceae, Clostridiaceae and Halomonadaceae.

Bifidobacterium longum subsp. infantis in experimental necrotizing enterocolitis: alterations in inflammation, innate immune response, and the microbiota.

BACKGROUND: Probiotics decrease the risk of necrotizing enterocolitis (NEC). We sought to determine the impact of Bifidobacterium longum subsp. infantis (B. infantis) in the established rat model of NEC. METHODS: Rat pups delivered 1 d prior to term gestation were assigned to one of three groups: dam fed (DF), formula fed (FF), or fed with formula supplemented with 5 × 10(6) CFU B. infantis per day (FF+Binf). Experimental pups were exposed to hypoxia and cold stress. Ileal tissue was examined for pathology and expression of inflammatory mediators, antimicrobial peptides, and goblet-cell products. Ceca were assessed for bacterial composition by analysis of the 16S rRNA sequence. RESULTS: Administration of B. infantis significantly reduced the incidence of NEC, decreased expression of Il6, Cxcl1, Tnfa, Il23, and iNOS, and decreased expression of the antimicrobial peptides Reg3b and Reg3g. There was significant microbial heterogeneity both within groups and between experiments. The cecal microbiota was not significantly different between the FF and FF+Binf groups. Bifidobacteria were not detected in the cecum in significant numbers. CONCLUSION: In the rat model, the inflammation associated with NEC was attenuated by administration of probiotic B. infantis. Dysbiosis was highly variable, precluding determination of the precise role of the microbiota in experimental NEC.

Prebiotic oligosaccharides in premature infants.

OBJECTIVE: The aim of the study was to determine the impact of increasing doses of 2 prebiotic oligosaccharides and of an "all-human diet" on the intestinal microbiota of premature infants. METHODS: Twelve premature infants receiving formula feedings were randomly assigned to receive either galacto-oligosaccharide (F+GOS) or a pooled concentrated donor human milk product containing human milk oligosaccharides (F+HMO) in increasing doses during a 5-week period. A second group of 15 premature infants received their mother's own milk fortified with either a concentrated donor human milk product (H+H) or a bovine powdered fortifier (H+B). Serial stool specimens from each infant were analyzed by terminal restriction fragment length polymorphism and quantitative polymerase chain reaction for bacterial composition. RESULTS: All of the infants studied had relatively low levels of bifidobacteria and no measurable Lactobacilli. Infants from the F+GOS and F+HMO groups demonstrated an increase in relative numbers of Clostridia with increasing doses. Compared with the H+B group, the infants in the F+HMO and the H+H groups showed an unexpected trend toward an increase in γ-Proteobacteria over time/dose. Principal coordinate analyses and Shannon diversity scores were not significantly different among the 4 groups. Infants in the H+H group received more antibiotics during the study period than those in the other groups. Two of the infants receiving GOS developed feeding intolerance. CONCLUSIONS: None of the prebiotic interventions resulted in significant increases in bifidobacteria compared with baseline specimens or the H+B group; however, many of the infants did not receive the highest doses of GOS and HMO, and antibiotic use in the H+H group was high.

Fecal shedding of SARS-CoV-2 in infants born to SARS-CoV-2 positive mothers: a pilot study.

Background: Fecal shedding of SARS-CoV-2 occurs during infection, particularly in pediatric populations. The gut microbiota are associated with resistance to enteric pathogens. COVID-19 is associated with alterations to the gut microbiome. We hypothesized that the gut microbiome of infants born to SARS-CoV-2+ mothers differs between infants with and without fecal shedding of the virus. Methods: We enrolled 10 infants born to SARS-CoV-2+ mothers. We used qPCR on fecal RNA to test for SARS-CoV-2 and 16S rRNA gene sequencing of the V4 region to assess the gut microbiome. Infant SARS-CoV-2 status from nasal swabs was abstracted from medical records. Results: Of the 10 included infants, nine were tested for SARS-CoV-2 by nasal swab with 1 testing positive. Four infants, including the nasal swab positive infant, had at least one sample with detectable levels of SARS-CoV-2 fecal shedding. Detection of both SARS-CoV-2 genes in feces was associated with increased gut alpha diversity compared to no detection by a linear mixed effects model (p < 0.001). Detection of both SARS-CoV-2 genes was associated with increased levels Erysipelotrichaceae, Lactobacillaceae, and Ruminococceae by MaAsLin2. Conclusion: Fecal shedding of SARS-CoV-2 occurs in infants who test negative on nasal swabs and is associated with differences in the gut microbiome.

A comparison of two probiotic strains of bifidobacteria in premature infants.

OBJECTIVE: To determine the impact of 2 probiotic bifidobacteria on the fecal microbiota of premature infants fed either human milk or formula. STUDY DESIGN: In the first of two phase 1 clinical trials, 12 premature infants receiving formula feedings were assigned randomly to receive either Bifidobacterium longum ssp infantis or Bifidobacterium animalis ssp lactis in increasing doses during a 5-week period. In the second, 9 premature infants receiving their mother's milk received each of the two bifidobacteria for 2 weeks separated by a 1-week washout period. Serial stool specimens from each infant were analyzed by terminal restriction fragment-length polymorphism and quantitative polymerase chain reaction for bacterial composition. RESULTS: Among the formula-fed infants, there was a greater increase in fecal bifidobacteria among infants receiving B infantis (Binf) than those receiving B lactis (Blac). This difference was most marked at a dose of 1.4 × 10(9) colony-forming units twice daily (P < .05). Bacterial diversity improved over dose/time in those infants receiving Binf. Among the human milk-fed infants, greater increases in fecal bifidobacteria and decreases in γ-Proteobacteria followed the administration of Binf than Blac. The B longum group (which includes Binf but not Blac) was the dominant bifidobacteria among the human milk-fed infants, regardless of the probiotic administered. CONCLUSIONS: Binf was more effective at colonizing the fecal microbiota than Blac in both formula-fed and human milk-fed premature infants. The combination of human milk plus Binf resulted in the greatest fecal levels of bifidobacteria.

Evidence of giant sulphur bacteria in Neoproterozoic phosphorites.

In situ phosphatization and reductive cell division have recently been discovered within the vacuolate sulphur-oxidizing bacteria. Here we show that certain Neoproterozoic Doushantuo Formation (about 600 million years bp) microfossils, including structures previously interpreted as the oldest known metazoan eggs and embryos, can be interpreted as giant vacuolate sulphur bacteria. Sulphur bacteria of the genus Thiomargarita have sizes and morphologies similar to those of many Doushantuo microfossils, including symmetrical cell clusters that result from multiple stages of reductive division in three planes. We also propose that Doushantuo phosphorite precipitation was mediated by these bacteria, as shown in modern Thiomargarita-associated phosphogenic sites, thus providing the taphonomic conditions that preserved other fossils known from the Doushantuo Formation.

Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities.

Bifidobacteria are intestinal anaerobes often associated with gut health. Specific bifidobacterial species are particularly common in the gastrointestinal tract of breast-fed infants. Current short read next-generation sequencing approaches to profile fecal microbial ecologies do not discriminate bifidobacteria to the species level. Here we describe a low-cost terminal restriction fragment length polymorphism (TRFLP) procedure to distinguish between the common infant-associated bifidobacterial species. An empirical database of TRF sizes was created from both common reference strains and well-identified isolates from infant feces. Species-specific quantitative PCR validated bifidobacterial-specific TRFLP profiles from infant feces. These results indicate that bifidobacterial-specific TRFLP is a useful method to monitor intestinal bifidobacterial populations from infant fecal samples. When used alongside next generation sequencing methods that detect broader population levels at lower resolution, this high-throughput, low-cost tool can help clarify the role of bifidobacteria in health and disease.

Lacto-N-tetraose, fucosylation, and secretor status are highly variable in human milk oligosaccharides from women delivering preterm.

Breast milk is the ideal nutrition for term infants but must be supplemented to provide adequate growth for most premature infants. Human milk oligosaccharides (HMOs) are remarkably abundant and diverse in breast milk and yet provide no nutritive value to the infant. HMOs appear to have at least two major functions: prebiotic activity (stimulation of the growth of commensal bacteria in the gut) and protection against pathogens. Investigations of HMOs in milk from women delivering preterm have been limited. We present the first detailed mass spectrometric analysis of the fucosylation and sialylation in HMOs in serial specimens of milk from 15 women delivering preterm and 7 women delivering at term using nanohigh performance liquid chromatography chip/time-of-flight mass spectrometry. A mixed-effects model with Levene's test was used for the statistical analyses. We find that lacto-N-tetraose, a core HMO, is both more abundant and more highly variable in the milk of women delivering preterm. Furthermore, fucosylation in preterm milk is not as well regulated as in term milk, resulting in higher within and between mother variation in women delivering preterm vs term. Of particular clinical interest, the α1,2-linked fucosylated oligosaccharide 2'-fucosyllactose, an indicator of secretor status, is not consistently present across lactation of several mothers that delivered preterm. The immaturity of HMO production does not appear to resolve over the time of lactation and may have relevance to the susceptibility of premature infants to necrotizing enterocolitis, late onset sepsis, and related neurodevelopmental impairments.

A comparison of bacterial colonization between nasogastric and orogastric enteral feeding tubes in infants in the neonatal intensive care unit.

OBJECTIVE: Feeding tubes harbor microbial contaminants; studies to date have not explored differences between orogastric (OG) and nasogastric (NG) tube biofilms. We sought to extend a previous analysis by comparing bacterial colonization by location (OG v NG) and by evaluating clinical factors that may affect tube bacterial populations. STUDY DESIGN: The pharyngeal segments of 41 infant feeding tubes (14 OG and 27 NG) from 41 infants were analyzed by next generation 16 S rRNA sequencing on the MiSeq platform. RESULTS: At the phylum level, Proteobacteria had the highest relative abundance of both OG and NG tubes. At the genus/species level, nine taxa differed significantly between OG and NG tubes. Alpha and beta diversity analyses showed significant differences between OG and NG tubes with relatively little contribution from clinical factors. CONCLUSION: The route of feeding tube insertion (oral vs nasal) had a greater impact on bacterial colonization than the assessed clinical factors.