Christopher J. Stewart

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim:  To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS).

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.

Gut microbiota in preterm infants: assessment and relevance to health and disease

In adults the microbial community of the gut (microbiota) influences a diverse range of health outcomes from obesity, diabetes, asthma and allergy to seemingly ‘remote’ diseases like Parkinsons disease.1 In preterm infants, establishment of the gut microbiota is also of importance for key morbidities like late onset sepsis (LOS) and necrotising enterocolitis (NEC), both significant causes of mortality.2 Many episodes of LOS are with gut derived organisms3 and changes in the intestinal barrier contribute to both LOS and NEC. The gut microbiota are key to developing barrier function, integrity, and mucosal and systemic immune function. They also ‘educate’ the gut associated lymphoid tissue, allowing the establishment of a ‘tolerant’ state between microbiota and the immune system, affecting intestinal function including tight junction structure and immune function.4–6 Patterns of initial colonisation affect host metabolic function: fat deposition, circulating leptin levels, and insulin resistance.6 In the preterm gut structural and immunological immaturity contribute to inflammatory necrosis and abnormal bacterial colonisation (dysbioses). This may result in decreased microbial diversity and an increased inflammatory response exacerbated by an immature innate immune response that increases the risk of diseases like NEC or LOS. An improved understanding of the microbiota of infants cared for in neonatal intensive care, and how this is affected by current practices may allow clinicians to promote more ‘healthy’ gut microbiota patterns, and may be associated with reductions in mortality and improvements in long term outcomes.7 ### What factors associated with preterm birth affect the gut microbiota? Early differences in delivery mode and care alter the development of the gut microbiota. Vaginally delivered term infants acquire their gut microbiota from maternal exposures (genital, stool, skin and breast milk flora) and the wider environment. In contrast, preterm infants are more commonly delivered by caesarean section, and have additional ‘risks and exposures’: antenatal and postnatal antibiotics, infection control measures …

Bacterial and fungal viability in the preterm gut: NEC and sepsis

Background and aims Evidence suggests that microbial communities in the preterm gut may influence the development of necrotising enterocolitis (NEC) and sepsis. Existing data often neglect fungi and whether bacteria were metabolically active or not. We sought to characterise the bacterial and fungal stool flora of preterm neonates and organism viability and evaluate any associations with NEC and sepsis. Patients 136 stools from 32 patients (<32 weeks gestation) were collected between birth and day 95. Seven infants developed NEC and 13 sepsis. Methods Stools were analysed by PCR-DGGE for assessment of the total bacterial and fungal communities by analysis of 16S rRNA and 28S rRNA, respectively. In 65 samples (25 infants), the viable (RNA) bacterial and fungal communities were analysed. Analyses were performed to examine the possible effects of demographic or treatment related factors and the development of NEC or sepsis. Results 80 (66 viable) bacterial species were identified overall and 12 fungal (none viable). Total bacterial communities significantly differed between healthy infants and those with NEC or sepsis, with Sphingomonas spp. significantly associated with NEC. Significant drivers of community structure differed based on either total or viable analysis. Antifungal prophylaxis was associated with altered bacterial community and a reduction in bacterial richness was observed in week 4, correlating with high antibiotic exposure. Conclusions Total and viable communities differ in preterm infants, and non-viable fungal species are present in infants on fungal prophylaxis. Exploration of viability and non-bacterial contributors to the total community may increase understanding of NEC and sepsis.

Lactoferrin: Antimicrobial activity and therapeutic potential

Lactoferrin is a highly conserved protein from an evolutionary perspective, with a wide range of roles related to protection from infection and promotion of nutritional status. Infection, malnutrition and intestinal pathologies are key inter-related problems, represent important threats to survival and are associated with adverse long-term health outcomes after preterm birth. Lactoferrin is available as a commercial extract from bovine milk and offers potential as a therapeutic intervention for preterm infants modulating infections and intestinal pathologies. In this review we explore the structure, direct antimicrobial effects, modification of host immune function and gastrointestinal effects of lactoferrin. Current trial data are reviewed, and research priorities and challenges identified and discussed.

The neonatal bowel microbiome in health and infection

Purpose of review In newborns, interactions between the host and the microbiome operate synergistically, modulating host immune function and shaping the microbiome. Next generation molecular sequencing methodologies in tandem with modeling complex communities allow insights into the role of the microbiome in health and disease states. Infection-related disease states in which dysbiosis is integral include late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), which still cause deaths and morbidity. Understanding microbiomic interactions may lead to alternative prevention, monitoring or treatment strategies, and modulation of long-term health outcomes especially in the preterm population. Recent studies have advanced understanding of the microbiome in NEC and LOS. Recent findings Mechanisms of host–microbiome interaction have been demonstrated. Patterns of microbiomic change in association with NEC and LOS have been observed, with community changes dominated by Proteobacteria and Firmicutes appearing to precede NEC, and very early microbiomic signatures influencing LOS. Data on viral and fungal elements are emerging. Summary Greater understanding of the neonatal bowel microbiome may allow tailored clinical practice and therapeutic intervention. Data handling and interpretation is challenging. Mechanistic studies of clinical interventions that affect the gut microbiome are important next steps.

Metabolomic and proteomic analysis of serum from preterm infants with necrotising entercolitis and late-onset sepsis

Background:Necrotising enterocolitis (NEC) and late-onset sepsis (LOS) are the leading causes of death among preterm infants in the developed world. This study aimed to explore the serum proteome and metabolome longitudinally in preterm infants with NEC or LOS, matched to controls.Methods:Nineteen patients (10 cases, 9 controls) were included. A sample 14 d prior to and following, as well as at disease diagnosis, was included for cases. Controls had serum matched at diagnosis for corresponding case. All samples (n = 39) underwent shotgun proteomic analysis, and 37 samples also underwent metabolomics analysis using ultra performance liquid chromatography–tandem mass spectrometry.Results:The proteomic and metabolomic profiles of serum were comparable between all infants. Eight proteins were associated with NEC and four proteins were associated with LOS. C-reactive protein was increased in all NEC patients at diagnosis.Conclusion:No single protein or metabolite was detected in all NEC or LOS cases which was absent from controls; however, several proteins were identified which were associated with disease status. The differing expression of these proteins between diseased infants potentially relates to differing pathophysiology of disease. Thus, it is unlikely a single biomarker exists for NEC and/or LOS.

Routine Use of Probiotics in Preterm Infants: Longitudinal Impact on the Microbiome and Metabolome.

Background: Probiotics are live microbial supplements that colonize the gut and potentially exert health benefit to the host. Objectives: We aimed to determine the impact of a probiotic (Infloran®: Lactobacillus acidophilus-NCIMB701748 and Bifidobacterium bifidum-ATCC15696) on the bacterial and metabolic function of the preterm gut while in the neonatal intensive care unit (NICU) and following discharge. Methods: Stool samples (n = 88) were collected before, during, and after probiotic intake from 7 patients, along with time-matched controls from 3 patients. Samples were also collected following discharge home from the NICU. Samples underwent bacterial profiling analysis by 16S rRNA gene sequencing and quantitative PCR (qPCR), as well as metabolomic profiling using liquid chromatography mass spectrometry. Results: Bacterial profiling showed greater Bifidobacterium (15.1%) and Lactobacillus (4.2%) during supplementation compared to the control group (4.0% and 0%, respectively). While Lactobacillus became reduced after the probiotic had been stopped, Bifidobacterium remained high following discharge, suggestive of successful colonisation. qPCR analysis showed a significant increase (p ≤ 0.01) in B. bifidum in infants who received probiotic treatment compared to controls, but no significant increase was observed for L. acidophilus (p = 0.153). Metabolite profiling showed clustering based on receiving probiotic or matched controls, with distinct metabolites associated with probiotic administration. Conclusions: Probiotic species successfully colonise the preterm gut, reducing the relative abundance of potentially pathogenic bacteria, and effecting gut functioning. Bifidobacterium (but not Lactobacillus) colonised the gut in the long term, suggesting the possibility that therapeutically administered probiotics may continue to exert important functional effects on gut microbial communities in early infancy.

Polymicrobial airway bacterial communities in adult bronchiectasis patients.

BackgroundChronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation.ResultsThe lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung.ConclusionsWe have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.

Gut Microbiota and Lifestyle Interventions in NAFLD

The human digestive system harbors a diverse and complex community of microorganisms that work in a symbiotic fashion with the host, contributing to metabolism, immune response and intestinal architecture. However, disruption of a stable and diverse community, termed “dysbiosis”, has been shown to have a profound impact upon health and disease. Emerging data demonstrate dysbiosis of the gut microbiota to be linked with non-alcoholic fatty liver disease (NAFLD). Although the exact mechanism(s) remain unknown, inflammation, damage to the intestinal membrane, and translocation of bacteria have all been suggested. Lifestyle intervention is undoubtedly effective at improving NAFLD, however, not all patients respond to these in the same manner. Furthermore, studies investigating the effects of lifestyle interventions on the gut microbiota in NAFLD patients are lacking. A deeper understanding of how different aspects of lifestyle (diet/nutrition/exercise) affect the host–microbiome interaction may allow for a more tailored approach to lifestyle intervention. With gut microbiota representing a key element of personalized medicine and nutrition, we review the effects of lifestyle interventions (diet and physical activity/exercise) on gut microbiota and how this impacts upon NAFLD prognosis.