Impact of probiotics on gut microbiome of extremely preterm or extremely low birthweight infants.

IMPACT: Meta-analysis of probiotic administration to very preterm or very low birthweight (VP/VLBW) infants shows reduced risk of necrotising enterocolitis (NEC). Separately reported outcomes for extremely preterm infants (<28 weeks) or extremely low birth weight infants (<1000 g) (EP/ELBW) are lacking meaning some clinicians do not administer probiotics to EP/ELBW infants despite their high risk of NEC. We present data showing the gut microbiome is impacted in EP/ELBW infants in a similar manner to VP/VLBW infants, suggesting that risk reduction for necrotising enterocolitis that is microbiome driven will also be seen in EP/ELBW infants, making probiotic administration beneficial.

Secretory immunoglobulin A in preterm infants: determination of normal values in breast milk and stool.

BACKGROUND: IgA and its secretory form sIgA impact protection from infection and necrotising enterocolitis but little is known about quantities in preterm mums own milk (MOM) or infant stool, onset of endogenous production in the preterm gut, and what affects these. METHODS: We measured by ELISA in MOM and stool from healthy preterm infants total IgA and sIgA longitudinally and additionally in MOM fresh, refrigerated, frozen, and after traversing feeding systems. RESULTS: In 42 MOM (median gestation 26 weeks), we showed total IgA levels and sIgA were highest in colostrum, fell over 3 weeks, and were not impacted by gestation. Median IgA values matched previous term studies (700 mcg/ml). In MOM recipients stool IgA was detected in the first week, at around 30% of MOM quantities. Formula fed infants did not have detectable stool IgA until the third week. Levels of IgA and sIgA were approximately halved by handling processes. CONCLUSIONS: MOM in the 3 weeks after preterm delivery contains the highest concentrations of IgA and sIgA. Endogenous production after preterm birth occurs from the 3 week meaning preterm infants are dependent on MOM for IgA which should be optimised. Routine NICU practices halve the amount available to the infant. IMPACT: (Secretory) Immunoglobulin A (IgA) is present in colostrum of maternal milk from infants as preterm as 23-24 weeks gestational age, falling over the first 3 weeks to steady levels similar to term. Gestation at birth does not impact (secretory) IgA levels in breast milk. IgA is present in very preterm infant stools from maternal milk fed infants from the first week of life, but not in formula milk fed preterm infants until week three, suggesting endogenous production from this point. Refrigeration, freezing, and feeding via plastic tubing approximately halved the amount of IgA available.

Use of omic technologies in early life gastrointestinal health and disease: from bench to bedside.

Introduction: At birth, the gastrointestinal (GI) tract is colonized by a complex community of microorganisms, forming the basis of the gut microbiome. The gut microbiome plays a fundamental role in host health, disorders of which can lead to an array of GI diseases, both short and long term. Pediatric GI diseases are responsible for significant morbidity and mortality, but many remain poorly understood. Recent advancements in high-throughput technologies have enabled deeper profiling of GI morbidities. Technologies, such as metagenomics, transcriptomics, proteomics and metabolomics, have already been used to identify associations with specific pathologies, and highlight an exciting area of research. However, since these diseases are often complex and multifactorial by nature, reliance on a single experimental approach may not capture the true biological complexity. Therefore, multi-omics aims to integrate singular omic data to further enhance our understanding of disease.Areas covered: This review will discuss and provide an overview of the main omic technologies that are used to study complex GI pathologies in early life.Expert opinion: Multi-omic technologies can help to unravel the complexities of several diseases during early life, aiding in biomarker discovery and enabling the development of novel therapeutics and augment predictive models.

Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Human milk microbiota, oligosaccharide profiles, and infant gut microbiome in preterm infants diagnosed with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a severe intestinal disease of very preterm infants with mother's own milk (MOM) providing protection, but the contribution of the MOM microbiota to NEC risk has not been explored. Here, we analyze MOM of 110 preterm infants (48 NEC, 62 control) in a cross-sectional study. Breast milk contains viable bacteria, but there is no significant difference in MOM microbiota between NEC and controls. Integrative analysis between MOM microbiota, human milk oligosaccharides (HMOs), and the infant gut microbiota shows positive correlations only between Acinetobacter in the infant gut and Acinetobacter and Staphylococcus in MOM. This study suggests that NEC protection from MOM is not modulated through the MOM microbiota. Thus, "'restoring" the MOM microbiota in donor human milk is unlikely to reduce NEC, and emphasis should instead focus on increasing fresh maternal human milk intake and researching different therapies for NEC prevention.

Mechanisms affecting the gut of preterm infants in enteral feeding trials: a nested cohort within a randomised controlled trial of lactoferrin.

OBJECTIVE: To determine the impact of supplemental bovine lactoferrin on the gut microbiome and metabolome of preterm infants. DESIGN: Cohort study nested within a randomised controlled trial (RCT). Infants across different trial arms were matched on several clinical variables. Bacteria and metabolite compositions of longitudinal stool and urine samples were analysed to investigate the impact of lactoferrin supplementation. SETTING: Thirteen UK hospitals participating in a RCT of lactoferrin. PATIENTS: 479 infants born <32 weeks' gestation between June 2016 and September 2017. RESULTS: 10 990 stool and 22 341 urine samples were collected. Analyses of gut microbiome (1304 stools, 201 infants), metabolites (171 stools, 83 infants; 225 urines, 90 infants) and volatile organic compounds (314 stools, 117 infants) were performed. Gut microbiome Shannon diversity at 34 weeks corrected age was not significantly different between infants in the lactoferrin (mean=1.24) or placebo (mean=1.06) groups (p=0.11). Lactoferrin receipt explained less than 1% variance in microbiome compositions between groups. Metabolomic analysis identified six discriminative features between trial groups. Hospital site (16%) and postnatal age (6%) explained the greatest variation in microbiome composition. CONCLUSIONS: This multiomic study identified minimal impacts of lactoferrin but much larger impacts of hospital site and postnatal age. This may be due to the specific lactoferrin product used, but more likely supports the findings of the RCT in which this study was nested, which showed no impact of lactoferrin on reducing rates of sepsis. Multisite mechanistic studies nested within RCTs are feasible and help inform trial interpretation and future trial design.

Necrotising enterocolitis, late-onset sepsis and mortality after routine probiotic introduction in the UK.

OBJECTIVE: To compare necrotising enterocolitis (NEC), late-onset sepsis (LOS), focal intestinal perforation (FIP) and mortality in infants from a single neonatal unit before and after probiotic introduction. DESIGN: Retrospective review of infants <32 weeks admitted January 2009-December 2012 (no probiotic) and January 2013-December 2017 (routine probiotics). Infants included were admitted before day 3, and not transferred out before day 3. NEC, LOS and FIP were defined with standard definitions. PATIENTS: 1061 infants were included, 509 preprobiotic and 552 postprobiotic. Median gestation, birth weight and antenatal steroid use did not differ, and proportions of extremely low birthweight infants were similar (37% and 41%). RESULTS: Overall unadjusted risk of NEC (9.2% (95% CI 7.1 to 12.1) vs 10.6% (95% CI 8.2 to 13.4), p=0.48), LOS (16.3% (95% CI 13.2 to 19.6) vs 14.1% (95% CI 11.5 to 17.4), p=0.37) and mortality (9.2% (95% CI 7.1 to 12.1) vs 9.7% (95% CI 7.6 to 12.6), p=0.76) did not differ, nor proportion of surgical NEC. In multiple logistic regression, accounting for gestation, birth weight, antenatal steroid, maternal milk, chorioamnionitis and sex, probiotic receipt was not significantly associated with NEC (adjusted OR (aOR) 1.08 (95% CI 0.71 to 1.68), p=0.73), LOS or mortality. In subgroup (645 infants) >28 weeks, aOR for NEC in the probiotic cohort was 0.42 (95% CI 0.2 to 0.99, p=0.047). FIP was more common in the probiotic cohort (OR 2.3 (95% CI 1.0 to 5.4), p=0.04), not significant in regression analysis (2.11 (95% CI 0.97 to 4.95), p=0.05). CONCLUSIONS: Probiotic use in this centre did not reduce overall mortality or rates of NEC, LOS or FIP but subgroup analysis identified NEC risk reduction in infants >28 weeks, and LOS reduction <28 weeks.

Strain-specific impacts of probiotics are a significant driver of gut microbiome development in very preterm infants.

The development of the gut microbiome from birth plays important roles in short- and long-term health, but factors influencing preterm gut microbiome development are poorly understood. In the present study, we use metagenomic sequencing to analyse 1,431 longitudinal stool samples from 123 very preterm infants (<32 weeks' gestation) who did not develop intestinal disease or sepsis over a study period of 10 years. During the study period, one cohort had no probiotic exposure whereas two cohorts were given different probiotic products: Infloran (Bifidobacterium bifidum and Lactobacillus acidophilus) or Labinic (B. bifidum, B. longum subsp. infantis and L. acidophilus). Mothers' own milk, breast milk fortifier, antibiotics and probiotics were significantly associated with the gut microbiome, with probiotics being the most significant factor. Probiotics drove microbiome transition into different preterm gut community types (PGCTs), each enriched in a different Bifidobacterium sp. and significantly associated with increased postnatal age. Functional analyses identified stool metabolites associated with PGCTs and, in preterm-derived organoids, sterile faecal supernatants impacted intestinal, organoid monolayer, gene expression in a PGCT-specific manner. The present study identifies specific influencers of gut microbiome development in very preterm infants, some of which overlap with those impacting term infants. The results highlight the importance of strain-specific differences in probiotic products and their impact on host interactions in the preterm gut.