ObjectiveNecrotising enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm infants. The underlying mechanisms are poorly understood: mother’s own breast milk (MOM) is protective, possibly relating to human milk oligosaccharide (HMO) and infant gut microbiome interplay. We investigated the interaction between HMO profiles and infant gut microbiome development and its association with NEC.DesignWe performed HMO profiling of MOM in a large cohort of infants with NEC (n=33) with matched controls (n=37). In a subset of 48 infants (14 with NEC), we also performed longitudinal metagenomic sequencing of infant stool (n=644).ResultsConcentration of a single HMO, disialyllacto-N-tetraose (DSLNT), was significantly lower in MOM received by infants with NEC compared with controls. A MOM threshold level of 241 nmol/mL had a sensitivity and specificity of 0.9 for NEC. Metagenomic sequencing before NEC onset showed significantly lower relative abundance of Bifidobacterium longum and higher relative abundance of Enterobacter cloacae in infants with NEC. Longitudinal development of the microbiome was also impacted by low MOM DSLNT associated with reduced transition into preterm gut community types dominated by Bifidobacterium spp and typically observed in older infants. Random forest analysis combining HMO and metagenome data before disease accurately classified 87.5% of infants as healthy or having NEC.ConclusionThese results demonstrate the importance of HMOs and gut microbiome in preterm infant health and disease. The findings offer potential targets for biomarker development, disease risk stratification and novel avenues for supplements that may prevent life-threatening disease.

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.

Necrotising enterocolitis (NEC) and sepsis are serious diseases of preterm infants that can result in feeding intolerance, the need for bowel resection, impaired physiological and neurological development, and high mortality rates. Neonatal healthcare improvements have allowed greater survival rates in preterm infants leading to increased numbers at risk of developing NEC and sepsis. Gut bacteria play a role in protection from or propensity to these conditions and have therefore, been studied extensively using targeted 16S rRNA gene sequencing methods. However, exact epidemiology of these conditions remain unknown and the role of the gut microbiota in NEC remains enigmatic. Many studies have confounding variables such as differing clinical intervention strategies or major methodological issues such as the inability of 16S rRNA gene sequencing methods to determine viable from non-viable taxa. Identification of viable community members is important to identify links between the microbiota and disease in the highly unstable preterm infant gut. This is especially important as remnant DNA is robust and persists in the sampling environment following cell death. Chelation of such DNA prevents downstream amplification and inclusion in microbiota characterisation. This study validates use of propidium monoazide (PMA), a DNA chelating agent that is excluded by an undamaged bacterial membrane, to reduce bias associated with 16S rRNA gene analysis of clinical stool samples. We aim to improve identification of the viable microbiota in order to increase the accuracy of clinical inferences made regarding the impact of the preterm gut microbiota on health and disease. Gut microbiota analysis was completed on stools from matched twins (n = 16) that received probiotics. Samples were treated with PMA, prior to bacterial DNA extraction. Meta-analysis highlighted a significant reduction in bacterial diversity in 68.8% of PMA treated samples as well as significantly reduced overall rare taxa abundance. Importantly, overall abundances of genera associated with protection from and propensity to NEC and sepsis such as: Bifidobacterium; Clostridium, and Staphylococcus sp. were significantly different following PMA-treatment. These results suggest non-viable cell exclusion by PMA-treatment reduces bias in gut microbiota analysis from which clinical inferences regarding patient susceptibility to NEC and sepsis are made.

Objectives: Microbial communities influencing health and disease are being increasingly studied in preterm neonates. There exists little data, however, detailing longitudinal microbial acquisition, especially in the most extremely preterm (<26 weeks’ gestation). This study aims to characterize the development of the microbiota in this previously under-represented cohort. Methods: Seven extremely preterm infant-mother dyads (mean gestation 23.6 weeks) were recruited from a single neonatal intensive care unit. Oral and endotracheal secretions, stool, and breast milk (n = 157 total), were collected over the first 60 days of life. Targeted 16S rRNA gene sequencing identified bacterial communities present. Results: Microbiota of all body sites were most similar immediately following birth and diverged longitudinally. Throughout the sampling period Escherichia, Enterococcus, Staphylococcus, and an Enterobacteriaceae were dominant and well dispersed across all sites. Temporal divergence of the stool from other microbiota was driven by decreasing diversity and significantly greater proportional abundance of Bifidobacteriaceae compared to other sites. Conclusions: Four taxa dominated all anatomical sampling sites. Rare taxa promoted dissimilarity. Cross-seeding between upstream communities and the stool was demonstrated, possibly relating to buccal colostrum/breast milk exposure and indwelling tubes. Given the importance of dysbiosis in health and disease of extremely preterm infants, better understanding of microbial acquisition within this context may be of clinical benefit.

Ionic liquids are salts used in a variety of industrial processes, and being relatively non-volatile, are proposed as environmentally-friendly replacements for existing volatile liquids. Methylimidazolium ionic liquids resist complete degradation in the environment, likely because the imidazolium moiety does not exist naturally in biological systems. However, there is limited data available regarding their mammalian effects in vivo. This study aimed to examine the effects of exposing mice separately to 2 different methylimidazolium ionic liquids (BMI and M8OI) through their addition to drinking water. Potential effects on key target organs-the liver and kidney-were examined, as well as the gut microbiome. Adult male mice were exposed to drinking water containing ionic liquids at a concentration of 440 mg/L for 18 weeks prior to examination of tissues, serum, urine and the gut microbiome. Histopathology was performed on tissues and clinical chemistry on serum for biomarkers of hepatic and renal injury. Bacterial DNA was isolated from the gut contents and subjected to targeted 16S rRNA sequencing. Mild hepatic and renal effects were limited to glycogen depletion and mild degenerative changes respectively. No hepatic or renal adverse effects were observed. In contrast, ionic liquid exposure altered gut microbial composition but not overall alpha diversity. Proportional abundance of Lachnospiraceae, Clostridia and Coriobacteriaceae spp. were significantly greater in ionic liquid-exposed mice, as were predicted KEGG functional pathways associated with xenobiotic and amino acid metabolism. Exposure to ionic liquids via drinking water therefore resulted in marked changes in the gut microbiome in mice prior to any overt pathological effects in target organs. Ionic liquids may be an emerging risk to health through their potential effects on the gut microbiome, which is implicated in the causes and/or severity of an array of chronic disease in humans.

Background: The objective of this study was to explore the clinical and microbiological outcomes associated with substituting inhaled aztreonam lysine for an intravenous antibiotic in the treatment of acute pulmonary exacerbations of CF.Methods: An open-label randomised crossover pilot trial was conducted at a UK CF centre among 16 adults with CF and P. aeruginosa infection. Median [IQR] age was 29.5 [24.5-32.5], mean ± SD forced expiratory volume in 1 second (FEV1) was 52.4 ± 14.7 % predicted. Over the course of two exacerbations, participants were randomised to sequentially receive 14 days of inhaled aztreonam lysine plus IV colistimethate (AZLI + IV), or dual IV antibiotics (IV + IV). Primary outcome was absolute change in % predicted FEV1. Other outcomes evaluated changes in quality of life, bacterial load and the lung microbiota. Results:The difference between mean change in lung function at day 14 between AZLI + IV and IV + IV was + 4.6% (95% CI 2.1-7.2, p = 0.002). The minimum clinically important difference of the Cystic Fibrosis Revised Questionnaire (CFQ-R) was achieved more frequently with AZLI + IV (10/12, 83.3%) than IV + IV (7/16, 43.8%), p = 0.05. No differences were observed for modulation of serum white cell count, C-reactive protein or sputum bacterial load. Microbiome compositional changes were observed with IV + IV (Bray-Curtis r 2 = 0.14, p = 0.02), but not AZLI + IV (r 2 = 0.03, p = 0.64). Conclusion:In adults with CF and P. aeruginosa infection experiencing an acute pulmonary exacerbation, AZLI + IV improved lung function and quality of life compared to the current standard treatment. These findings support the need for larger definitive trials of inhaled antibiotics in the acute setting.

Dietary periodization is employed to improve endurance exercise performance but may impact on gut microbial communities. Bacteriophage are implicated in bacterial cell homeostasis and have been identified as biomarkers of disequilibrium in the gut ecosystem possibly brought about through dietary periodization.

The fecal metabolome in early life has seldom been studied. We investigated its evolution in pre-term babies during their first weeks of life. Multiple (n = 152) stool samples were studied from 51 babies, all <32 weeks gestation. Volatile organic compounds (VOCs) were analyzed by headspace solid phase microextraction gas chromatography mass spectrometry. Data were interpreted using Automated Mass Spectral Deconvolution System (AMDIS) with the National Institute of Standards and Technology (NIST) reference library. Statistical analysis was based on linear mixed modelling, the number of VOCs increased over time; a rise was mainly observed between day 5 and day 10. The shift at day 5 was associated with products of branched-chain fatty acids. Prior to this, the metabolome was dominated by aldehydes and acetic acid. Caesarean delivery showed a modest association with molecules of fungal origin. This study shows how the metabolome changes in early life in pre-term babies. The shift in the metabolome 5 days after delivery coincides with the establishment of enteral feeding and the transition from meconium to feces. Great diversity of metabolites was associated with being fed greater volumes of milk.