Berrington, Janet E.a; Stewart, Christopher J.b; Cummings, Stephen P.b; Embleton, Nicholas D.a
The complex microbial community (microbiome) in the human gut is viewed as a superorgan involved in many aspects of health and disease in which the microbiome exerts metabolic, nutritional, and immunological effects on the host . In healthy, vaginally delivered, term newborns (HVDTNB) the gut and adaptive immune system tolerate and regulate this community; the microbiome in turn sculpts gut immune and metabolic function . The establishment of the microbiome proceeds predictably, reflecting the coevolution with their host over millennia [3,4]. However, this dynamic process is readily disrupted, especially by events related to prematurity . Such disruption, termed dysbiosis, is implicated in development of late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), which are both major causes of mortality and morbidity . Therapeutic manipulation and even microbiome transplantation are possible but the mechanistic interactions involved are complex and poorly understood. This review summarizes how advances in molecular technologies can offer mechanistic insights into host–microbiome interactions and reviews recent work suggesting a microbiome pattern representative of health and changes that precede LOS and NEC. We review progress toward our understanding of the functional contribution of bacterial community members, and the significance of nonbacterial organisms (fungi, viruses, and archaea). Insights from interventions intended to manipulate the microbiome are considered and a brief examination of residual research needs and priorities are outlined.
Over time the gut microbiota changes in constituent members, relative abundance, gene expression, and metabolic activity. Measuring this dynamic complexity poses significant challenges: analysis typically involves passed stool (rarely ileal fluid or tissue) and is thus limited by spontaneous stooling (which occurs less frequently when infants are ill) and reflects alterations that are historical and dependent on transit times. Analyzing and presenting such complex dynamic data sets also present many challenges.
Culture independent analysis exploits the presence of the ubiquitous and evolutionarily conserved 16S ribosomal RNA (16S rRNA) gene, by which prokaryotic taxa can be differentiated. Molecular analyses are facilitated by recent work showing that once sampled, the stool microbiome is stable at room temperature for several weeks . Samples undergo DNA extraction before universal PCR primers allow amplification of intervening hypervariable regions. These amplicons then require differentiating into sufficiently similar groups to be classified together into an operational taxonomic unit (OTU). Different techniques have been used and continue to develop (Fig. 1[8▪]). Bacterial classification is determined by using open access sequence databases.
Methodologies for nonbacterial constituents are less robust or well developed  but fungal, archaeal, protozoal, and viral microbiomic studies now exist. The functional capacity of the microbiome is also increasingly acknowledged: metabolomic and proteomic techniques offer insight into functional microbiomic changes associated with disease .
Next-generation sequencing techniques (Fig. 1) generate large and complex datasets. Indeed, the subsequent bioinformatic and statistical analysis of these datasets have become the bottleneck for microbial community analyses . Communities are described in terms of richness (their constituents), evenness (dominance by particular OTUs), and diversity (a combination of richness and evenness). Ordination analyses and constrained or redundancy analyses allow exploration of potential mediators of structure and changes , approaches that are particularly valuable when attempting to relate microbiomic data to the patient's demographic and clinical details. New methods of handling and interpreting such complex datasets are in evolution [13▪▪].
The neonatal period uniquely challenges the host: pioneering organisms are encountered by naïve host gut defences, educate and modulate immune responses, and thus regulate the microbiome development. The bacterial community structure and host defences modulate gut endothelial cells, modifying function, enhancing protection and maintaining barrier function. Careful regulation of pro- and anti-inflammatory effects is needed to maintain health . Recent mechanistic insights are outlined below.
Competitive evolutionary strategies
Studies in a murine model suggest direct pathogen control occurs through commensal bacteria controlling virulence factor expression in pathogenic bacteria, and by competition modulated through shared carbohydrate requirements [15▪]. Environmental acidification by Bifidobacteria as a means of controlling Escherichia coli has also been demonstrated, a mechanism of potential import for probiotic reduction of incidence of NEC .
The traditional top-down hypothesis of a breach of barrier integrity, with breakdown in endothelial tight junctions leading to loss of regulation of factors intrinsic to a balanced pro- and anti-inflammatory response has recently been challenged. An alternative hypothesis, that of Paneth cell dysregulation, has been proposed that challenges many traditional assumptions [17▪▪]. The antibacterial lectin RegIII gamma has been shown to modulate both total bacterial load and contact of luminal bacteria with host epithelium .
Several mechanisms have recently been demonstrated through which intraluminal bacteria modify host responses. Initial neonatal bacterial exposure within the gut leads to activation of Toll-like receptor (TLR)-4, which has been shown to activate an inhibitory loop through the expression of miR-146a (a small noncoding mRNA), affecting the response to TLR agonist [19▪▪]. Some Bifidobacterium bifidum can generate a proinflammatory Th17 immune response (involved in protection against mucosal infection), and functional Treg lymphocytes with plasticity for either secreting IL-17 or suppressing activation of the immune system, depending on the external stimuli . Polysaccharide A (PSA), a specific symbiosis factor produced by the commensal Bacteroides fragilis, uses TLR-2 receptor activation to activate Foxp3+ Treg cells to promote tolerance in a mouse model. PSA-lacking B. fragilis cannot promote this response, clearly demonstrating how specific bacterial products of symbionts manipulate host immune responses . Butyrate, a short-chain fatty acid (SCFA) produced by commensal microorganisms during starch fermentation, has also been shown to induce extrathalamic Treg differentiation [22▪,23▪].
DEFINING THE HEALTHY NEONATAL GUT MICROBIOME
A healthy gut microbiome is ‘the intestinal microbial community that assists the host to maintain a healthy status under certain environmental conditions’ . If this could be defined for the neonate, a comparison with disease states could be made, allowing insights into pathogenesis or guiding preventive or treatment options.
The full-term vaginally delivered breast-fed (FTVDBF) infant is considered ideal from a microbiomic perspective and was initially studied in 14 infants . The more comprehensive Norwegian Microflora Study (NOMIC) enrolled 246 term infants at day 4, 10, 30, and 120 of life and showed links between early colonization patterns and later growth patterns [26▪▪]. Specifically, Bacteroides appeared protective against obesity . Three phyla – Bacteroidetes, Proteobacteria, and Firmicutes – exert the greatest effects on community dynamics [4,28]. Bifidobacterium appears specifically important to the neonate: six species in breast milk showed properties expected of commercial probiotics , and specific probiotic formulations for preterm infants could be formulated with this knowledge .
Neonates born preterm are exposed to potentially detrimental factors in the development of the healthy microbiome, including prelabour rupture of membranes, caesarean delivery, maternal and neonatal antibiotic intervention, and lack of breast-feeding. Previous studies, although limited, showed reduced diversity and relative instability  (Fig. 2). Recently Arboleya et al.[32▪] studied 21 preterm infants (32 weeks mean gestation) and 20 FTVDBF infants: the aim was to establish how differently 18 microbial groups were represented, and SCFAs were also measured reflecting microbiome metabolic activity. Increased facultative anaerobic microorganisms including Enterobacteriaceae, Enterococcaceae, and Lactobacillus and fewer anaerobes, including Bifidobacterium, Bacteroidetes, and Atopobium, were seen in the preterm cohort. The authors hypothesize that this delay in aerobic establishment may delay immune maturation in preterm neonates. Further work in an entirely preterm cohort exploring the development of the gut microbiota from multiple births also found Bifidobacterium abundance to be low [33▪]. Lower levels of naturally occurring probiotic species in preterm infants are important, given the possibility of supplementing these for the prevention of NEC .
SPECIFIC INFECTIOUS DISEASES
Dysbiosis appears pivotal to both LOS and NEC (Fig. 2). Patterns are beginning to emerge that appear associated, but causality is not proven.
NEC, an important neonatal disorder, is increasingly recognized as a spectrum of disease culminating in a final common pathway.
Is there a signature microbial pattern associated with NEC? If so, does it predate NEC onset to allow early diagnosis or modification of practice to improve outcomes?
Recent studies addressing these questions are shown in Table 1, highlighting populations, molecular techniques employed, and key findings. A pyrosequencing study of nine preterm infants with NEC sampled before NEC diagnosis and within 72 h of diagnosis identified a bloom in Proteobacteria and a decrease in Firmicutes between sampling, not seen in healthy controls . A potential ‘signature’ seen more frequently in NEC was identified as grouping to the Enterobacteriaceae family, but did not match any sequence at greater than 97%. Likewise, a case report of a single preterm infant (25 weeks gestation) who developed lethal NEC on day 48 with a dramatic increase in Proteobacteria, specifically Klebsiella, 2 days before. Proteobacteria contributed greater than 97% of phyla compared with less than 50% before. Ileal fluid and mucosal samples also identified the same pattern [36▪]. Jenke [37▪▪] identified an increase in E. coli preceding NEC in comparison both to earlier samples from the same individual and to healthy preterm infants. Fecal inflammatory markers were also studied: S100A12 and hBD2 were seen to correlate with total bacterial counts and E. coli, linking microbiomic changes and host immune responses. Two potentially distinct patterns of dysbiosis were identified by Morrow et al.[38▪▪] in 11 NEC infants: an early presentation (n = 4) dominated by Firmicutes, and a later presentation dominated by Proteobacteria. In our own cohort of infants less than 32 weeks gestation, distinct microbiomic differences between NEC and/or LOS compared with healthy infants were observed [33▪]. Detailed examination of twins discordant for NEC demonstrated a reduction in diversity and increasing dominance of Escherichia (since identified as E. coli) preceding NEC [39▪]. Preterm infants, whose exposure to lipopolysaccharide (LPS) from nosocomial bacteria is modified by neonatal intensive care practices, may be less tolerant when they do meet these bacteria and mount a resultant excessive inflammatory response. Interestingly, a Danish group did not identify microbiomic shifts in association with NEC using molecular analysis, although the timing of the sampling in relation to NEC development in this cohort is not described, and is clearly key . This group also analyzed small intestine specimens resected surgically in infants with NEC, demonstrating technical feasibility, but no specific profiles were identified associated with NEC .
Increasingly the role of nonbacterial organisms is being recognized in disease etiology.
Palmer  identified archaea in infants gut, but considerably less frequently and more variably than fungi or bacteria, appearing only transiently in the first few weeks of life. In inflammatory bowel disease (IBD), the presence of methanogenic archaea in the human gut was indicative of a healthy microbiota, with reduced methanogen presence in individuals with IBD : their absence from neonates may thus be important and mark dysbiosis.
Viruses may contribute to NEC by direct host viral infection or by phage infection of a microbiomic constituent. Torovirus  and astrovirus  have been previously linked to NEC onset, but recently there has been a resurgence of interest in cytomegalovirus (CMV). CMV was reported in association with fulminant lethal NEC in a 25-week-gestation infant: post-mortem inclusion bodies prompted recognition of CMV in the intestine [36▪]. An associated bloom in Proteobacteria was noted: CMV may have contributed to direct mucosal injury (seen in patients with IBD and CMV) or have promoted a microbiomic shift that heralded NEC. Deep sequencing of viral RNAs and DNAs revealed that many viruses in the infant gut are bacteriophages, although less diversity is seen in infants . Phages may influence microbiome composition by infecting and lysing specific bacterial hosts, allowing another bacterial strain the opportunity to become abundant . Although identified as present in the neonatal gut they have not been studied specifically in the context of NEC or LOS .
LaTuga et al. extended microbiomic analysis beyond the bacterial elements in their study of 11 infants less than 1000 g in their first month, five of whom had coagulase-negative staphylococci infection and two of whom developed NEC (but only one within the sampling window). They identified fungal species including several Candida species despite nystatin prophylaxis, a finding confirmed by Stewart et al.[49▪] who identified 12 fungal species (all nonviable) in their cohort who received fluconazole prophylaxis. The role of fungi in NEC pathogenesis requires further elucidation.
There are many parallels between LOS and NEC. Gut dysbiosis with a preponderance of Proteobacteria and Firmicutes abundance has been noted in neonates with LOS. Altered barrier and immune function and imbalance between pro- and anti-inflammatory responses have also been implicated. Mai et al.[42▪] analyzed 10 preterminfants with LOS and 18 preterm controls, showing association between microbiomic signatures in the two weeks before diagnosis of LOS but interestingly not at diagnosis. A high-resolution study of six preterm infants showed differences in microbial patterns in the first weeks of life in infants at high risk of sepsis: less diversity and a preponderance of staphylococci [41▪]. Archaea, viruses, and fungi in the gut may also influence LOS, but as with NEC, studies to date do not adequately analyze these domains in the pathogenesis of sepsis.
Interventions that affect the microbiome affect health: prolonged early postnatal antibiotics increase the risk of NEC , oral antibiotics appear to reduce it , probiotics can reduce NEC , lactoferrin appears to reduce LOS and possibly NEC [53,54]; however, mechanistic understanding for these findings is lacking.
However, clinical interventions could theoretically be manipulated and tailored to engineer a gut microbiota reflective of an FTVDBF infant. Breast milk contains complex immune-protective and growth factors, bioactive immune-modulatory cells and other immunonutrients including amino acids, fatty acids, lysozyme, lactoferrin, minerals, and metals such as zinc, and prebiotic oligosaccharides as well as live bacteria. It seems unlikely that artificial supplements could ever replicate the complexity of own mothers’ breast milk. Manufacturing complex combination supplements is attractive, but a better understanding is needed before this would be feasible. Few clinical trials in neonates have attempted mechanistic evaluations: few probiotic studies assess the microbiome – it is hypothesized but unproven that probiotics result in a reduction in the growth of potential pathogens including enterobacteria, enterococci, and clostridia . A serendipitous increase in Lactobacillus spp. in preterm infants was associated with 1% lactulose added to feeds, potentially attributable to its prebiotic effects . Lactoferrin, a molecule with many mechanistic effects on the microbiome, has not been subject to detailed exploration of the effects on the preterm infant microbiome .
Understanding the neonatal microbiome may hold the key to preventing LOS and NEC. Although patterns are emerging, the causal pathway is unclear. Where interventions do reduce morbidities (e.g., probiotics), the mechanisms are poorly understood, and key questions remain unanswered (Fig. 3). Mechanistic evaluation in the context of controlled clinical trials would help elucidate this, and may provide insight that allows improved prevention, diagnosis, and treatment. The inclusion of microbiomic assessment, including bacterial and nonbacterial elements and noninvasive sample collection, in future clinical trials of microbiomic modulation deserves further attention. This could include trials of feeding, probiotics, and enteral or immunonutrient supplements. Key unknowns about host response should also be addressed. Little is known about gut immune tissue responses to NEC in humans, yet many infants have resection of gut as part of their treatment, and immunohistochemistry techniques now allow elucidation of these aspects.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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