OUP user menu

Intestinal microbiota development in the premature neonate: establishment of a lasting commensal relationship?

Maka Mshvildadze, Josef Neu, Volker Mai
DOI: http://dx.doi.org/10.1111/j.1753-4887.2008.00119.x 658-663 First published online: 1 November 2008

Abstract

The gastrointestinal tract of premature infants is a highly fragile organ due to numerous developmental immaturities. Exposure to luminal microbes in the first several weeks of these infants' lives may play a significant role in the development of short-term disease and may have profound effects on long-term health. New non-culture-based techniques are providing exciting new insights into how the intestinal microbiota of premature infants develops and relates to health. A brief summary of recent research in this area is presented, which may be adapted to nutritional strategies for disease prevention.

  • intestine
  • microbiome
  • microbiota
  • necrotizing enterocolitis
  • premature infant

INTRODUCTION

The gastrointestinal tract (GI) of the premature infant has a large but fragile surface area covered by a thin monolayer of epithelial cells that overlies a highly immunoreactive submucosa. Interactions in the lumen among microbes, nutrients, and the intestinal mucosa can range from a healthy homeostasis to an uncontrolled systemic inflammatory response syndrome (SIRS) that leads to multiple organ system failure and death. Recent advances in molecular microbiota analytic methodology that stem from advances in high-throughput sequencing technology have provided us with the tools to determine the GI microbiota in great depth, including the nearly 70% of microbes in the intestine that cannot be cultured by current methodology.1,2 Application of these techniques to derive a better understanding of the developing intestinal ecosystem in the premature neonate may hold the key to understanding and eventually preventing several important diseases including necrotizing enterocolitis (NEC) and late-onset neonatal sepsis that may be acquired via translocation through the GI tract.

Summarized here are some of the recent developments in non-culture-based techniques of intestinal microbial analysis and recently acquired knowledge about how the premature neonates' intestinal microbiota interacts with the developing intestine and the luminal environment. Also described are host/intestinal microbiota interactions between the immune and other organ systems of the developing infant and how they might be modified by environmental factors such as diet, mode of delivery, and pharmacologic interventions during this highly vulnerable period of life. Adequate knowledge of the types of microorganisms as well as the events that influence the timing of colonization may provide opportunities to modulate the microbiota when needed to improve health.

TECHNIQUES FOR MICROBIOME ANALYSIS

In recent years molecular 16S-based techniques have been developed for efficient detection of different groups of bacteria in fecal samples without the need for culture, thus greatly facilitating the study of GI tract microbial ecology. The 16S rRNA gene contains highly conserved sequence domains interspersed with more variable regions. Consequently, comparative analyses of these variable rRNA sequences can identify so-called signature sequence motifs that can help identify different bacteria subpopulations. Table 1 summarizes some of these techniques and provides indications for their utilization. Following DNA extraction from fecal samples, amplification of the targeted 16S rRNA genes can be achieved with universal PCR primers capable of efficient annealing to rRNA genes from most bacteria; alternatively, specific primers designed to amplify rRNA genes from a particular bacterial group of interest can be used. Sanger sequencing-based 16S rRNA analysis has long been the gold standard for analyzing complex microbial communities. In this method, PCR products obtained using universal primers are cloned and inserts from individual clones are sequenced. Based on the degree of nucleotide similarity (usually between 95% and 99% similarity) sequences are then separated into operational taxonomic units (OTU) that form the basis for comparisons between samples. Due to the high cost of sequencing more than a few hundred clones, comprehensive coverage of all OTUs present is not achieved in highly complex samples, such as stool or soil samples. More recently, a new technique called immersion-based pyrosequencing (454 Life Sciences, Branford, CT, USA) has helped to overcome this limitation, as up to 400K sequences can now be obtained in a single run for a cost of less than US$0.05/sequence. Using a bar-coding approach, 100 different pools of PCR products (from 100 different samples) can be sequenced at a depth of 4000 sequences each. The bioinformatics tools required to efficiently analyze such large data sets are demanding and currently under development.3 The 16S rRNA sequencing approach becomes quantitative once sequencing is performed at sufficient depth to limit the impact of sampling variation and full saturation is reached (i.e., all OTUs present are detected).

View this table:
Table 1

Summary of techniques used for identifying intestinal microbiota.

Quantitative PCR (qPCR) and fluorescent in situ hybridization (FISH) are established methods to determine the total amounts or proportions of bacterial groups of interest in a complex microbial community. Other approaches that include DGGE/TGGE, RFLP, and ARISA allow for an efficient initial profiling of microbiota composition that can be applied to large studies. One of the more frequently utilized 16S rRNA gene-based profiling techniques is PCR-DGGE, a technique in which DNA is isolated from fecal samples and amplified by PCR using conserved 16S rDNA bacteria-domain primers. Total bacterial DNA from fecal samples is extracted and a hypervariable 16S rRNA region is amplified. One of the primers contains a “G-C clamp” that prevents the two DNA strands from dissociating completely, even under highly denaturing conditions. The resulting mixture of 16S rDNA fragments is subjected to a denaturing gradient, established in a polyacrylamide gel with urea and formamide, to separate the fragments and generate a “genetic fingerprint” of the microbiota present. Although all PCR products are of approximately equal size, individual amplicons stop migrating as the double-stranded products denature, which occurs according to their specific sequence-based denaturing characteristics (largely determined by G-C content). This approach allows separation of sequences based on denaturing characteristics that are thought to correspond to the different microbial groups within the sample. However, due to the need for target DNA amplification, most of the 16S-based approaches suffer from a PCR bias that has the potential to result in misrepresentation of true microbiota composition. Fluorescent in situ hybridization (FISH) doesn't include a PCR step, but it suffers from its own hybridization biases. Although the extent of these biases has not been well studied, it is apparent that a standardization of microbiota analysis methodology would greatly improve our ability to compare results across studies.

LUMINAL MICROBIOTA-INTESTINAL MUCOSA INTERACTIONS

The microbiota of the adult human is found primarily in the distal intestine and consists of >1013 microorganisms, likely comprising more than 500 species. The intestinal microbiota has a profound effect on nutritional status, development of the GI tract, and maintenance of mucosal surface integrity.46

Preterm infants have several immaturities of the GI tract.7,8 These, in combination with a highly reactive mucosa,9 may predispose to SIRS, which can ultimately lead to multiple organ dysfunction syndromes. Based on the profound immunoreactivity of the GI submucosa and liver, along with the exquisite sensitivity of the neonatal intestinal surface to translocation of inflammatory agents, it is likely that intestinal microbiota play a critical role in the prevention or pathogenesis of not only neonatal intestinal diseases such as NEC but also cholestatic liver failure, chronic lung disease, and central nervous system problems such as periventricular leukomalacia, which have been associated with systemic inflammation.10

Early development of the microbiota

The GI tract of a normal fetus is sterile. During the birth process and rapidly thereafter, microbes from the mother and the surrounding environment colonize the gastrointestinal tract of the infant, eventually leading to a dense and diverse bacterial community.11 This colonization is a complex process that likely is influenced by host/microbe interactions as well as internal and external exposures such as type of feeding (breast versus formula) and mode of delivery (caesarian versus vaginal).12,13 Changes in intestinal microbiota composition/activities and host cell gene expression affect the developing intestine and can affect the efficiency of nutrient uptake. Reduction of normal commensal microbiota diversity due to overgrowth by infectious agents or antibiotic treatment may thus interfere with the availability of critical nutrients and impair beneficial stimulation of GI mucosal development and the innate and adaptive immune responses.14

With core microbiome formation being dependent on exposure to the microbes that first colonize the GI tract, the establishment of a “healthy” microbiota in the first several days after birth is likely to be critical for normal development. Thus, factors that can affect microbe exposure such as mode of delivery, use of antibiotics, living in a neonatal intensive care unit with a potentially high load of pathogens, exposure to the mother's oral and skin microbiota, and breast milk or formula ingestion, are all likely to play important roles. The core microbiota, once developed in the individual, may be difficult to eradicate or modify. Furthermore, the initial core microbiota may affect the cascade of colonization by other microbes that results in full diversity,15 which may also play a large role in overall future health. Whether the infant is born prematurely and requires intensive care or is a term infant who is born without special needs could markedly affect the development of the intestinal microbial core.

Although several studies have monitored the bacterial communities in preterm infants, our picture of the microbiota still remains limited; culture-based techniques have been mainly used. In the adult human gut, 60–80% of the total microbiota couldn't be cultivated.16 To determine whether noncultured bacteria represents an important part of the community in premature babies' intestinal ecosystems, Magne et al.17 used 16S rRNA genes and PCR-TTGE profiling of 288 clones obtained from the fecal samples of 16 preterm infants. These were classified into 25 molecular species. The mean number of molecular species per infant was 3.25 and ranged from one to eight. The researchers found high interindividual variability. The main bacterial groups encountered belonged to the Enterobacteriaceae family and the genera Enterococcus, Streptococcus, and Staphylococcus. The preterm infants were colonized by anaerobes and only four bifidobacteria. The researchers did not determine the relative impacts of delivery mode, sex, gestational age, birth weight, age at sampling, feeding modes, and antibiotic therapies, but they assessed the global diversity of the fecal microbiota. They concluded that species diversity was low and interindividual variability was high in the feces of preterm infants, as revealed by sequences of 16S rRNA genes and PCR- temporal temperature gradient gel electrophoresis profiles. The intestinal ecosystem of these preterm infants had no typical characteristic. These data are in disagreement with an earlier study based on the DGGE profiles.11 In the latter study, DGGE profiles for all preterm infants were very simple at birth, diversity increased over time, and all preterm infants harbored a similar bacterial composition. This was also in accord with a DGGE-based study in which bands corresponding to Escherichia coli, Enterococcus sp., and Klebsiella pneumoniae were the most frequently encountered bacterial species in profiles.11

In another study using culture and molecular methods for analyzing bifidobacterial colonization in 52 infants born at a gestational age of 30–35 weeks,18 it was shown that colonization by bifidobacteria was not affected by birth weight, mode of delivery, antibiotics given to the mother and infant, or type of feeding, but gestational age at birth was a significant condition for colonization by bifidobacteria. The threshold birth age of 32.9 weeks may at least partially condition gut colonization by bifidobacteria. However, this study did not involve premature babies younger than 30 weeks and the development of bifidobacteria and evolution of GI microbiota in this group still remains unknown.

Term versus preterm infants

Favier et al.19 used PCR-DGGE to study the development of infant flora up to the age of 12 months. They were able to show that band patterns during the first days of life were very simple, but they became more complex over time.

In a step toward greater systematic investigation of babies born at term, Palmer et al.20 were the first to use microarray techniques to detect and quantify the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria; this was done along with sequencing of cloned libraries of PCR-amplified SSU rDNA to profile the microbial communities in 14 healthy full-term infants during the first year after birth. To investigate possible origins of the infant microbiota, the researchers also profiled vaginal and milk samples from most of the mothers as well as stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns from a set of dizygotic twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.

The intestinal bacterial colonization of preterm neonates differs from that of term infants both temporally and qualitatively. In general, studies investigating premature infants' intestinal bacterial colonization with beneficial bacteria suggest this is delayed and the number of potentially pathogenic bacteria is very high.21 It is not known whether prematurity itself may influence the intestinal establishment of microbiota. Many preterm infants require intensive care procedures early in life, which may be one of the major factors determining intestinal microbiota development. Many preterm infants receive antibiotics for ill-defined criteria, which are thought to have an influence of bacterial colonization,22 but their long-term effects have not been rigorously investigated.

In some studies, bifidobacteria were not detected directly after birth but they were detected increasingly over time.23 In two studies, bifidobacteria were rarely found during the whole study period.24,25 In only one study was a high number of bifidobacteria found directly after birth.26 A recent review21 summarized that preterm infants are colonized by low numbers of beneficial bacteria, such as bifidobacteria and lactobacilli, high numbers of potentially pathogenic bacteria, such as enterobacteria and E. coli, and high numbers of Bacteroides species, enterococci, and streptococci. As for pathogenic bacteria, high numbers of clostridia, staphylococci, and Klebsiella species are found in the intestinal bacterial microbiota of preterm infants while the numbers of Pseudomonas species are low.

Formula versus breastfeeding

The effect of diet on the composition of the infant GI microbiota is controversial. Several studies have found a lower abundance of bifidobacteria and a higher abundance of aerobic bacteria in the GI microbiota of formula-fed infants relative to breastfed infants,19,27 yet other reports have found no such difference.28,29 Another study compared the intestinal microbiota of breastfed versus formula-fed preterm infants using 16S gel techniques, and showed that breastfed infants have a more diverse bacterial population than formula-fed infants with lower numbers of pathogenic microorganisms.25 Studies have also shown that microbes and their cellular products are present in human mothers' milk, which may represent translocation through her GI tract and bloodstream into the breast milk.30 The implications of this remain unclear, but it is speculated that they may offer a protective effect for the neonate by interaction with intestinal cellular receptors that modulate the inflammatory response. Additional comprehensive non-culture-based analyses are needed to provide the critical data required to evaluate differences in the intestinal microbiota of formula-fed versus breastfed premature infants. This may provide guidance in terms of manipulations that would lead to establishment and maintenance of a healthy microbiota.

Mode of delivery

Mode of delivery has frequently been cited as one of the key factors that shape the microbiota of infants.28,31 The GI microbiota of infants delivered by caesarean section have been reported to differ from those of infants delivered vaginally, both in the timing and composition of colonization.2834 More recent studies applying newer technologies support this concept further.12,13 However, the relative importance of mode of delivery on GI microbiota composition and subsequent health is unclear.

Antibiotics

Little is known about how antibiotics alter the establishment of GI microbiota in premature infants. Several studies support that colonization with bacteria, especially lactobacilli is delayed in infants receiving antibiotics after birth.2426 However, most of these studies used conventional cultivation techniques.

In a study evaluating the response of intestinal development in rodents after antibiotic administration, Schumann et al.35 showed that neonatal antibiotic treatment alters the establishment of an efficient barrier to luminal antigens and bacterial colonization. Daily intragastric gavage of amoxicillin resulted in the near complete eradication of lactobacillus in the whole intestine and in a drastic reduction of colonic total aerobic and anaerobic bacteria, in particular enterobacteria and enterococci.35 This had marked effects on developmental gene expression, including those affecting development of the immune response.

Compared to studies in human and animal neonates, more information is available in adult studies about the antibiotic treatment and altered GI microbiota. Jarnberg et al.36 evaluated the long-term ecological impact of antibiotic administration on the human intestinal microbiota using molecular techniques after 7 days of clindamycin treatment. Two years after the treatment, highly significant disturbances in the bacterial community persisted throughout the sampling period. In particular, a sharp decline in the clonal diversity of Bacteroides isolates, as assessed by repetitive sequence-based PCR (rep-PCR), and long-term persistence of highly resistant clones were found as a direct response to the antibiotic exposure. A large decrease in the collective number of Bacteroides clone types from all individuals in the exposed group was seen on the last day of clindamycin exposure (day 7). The Bacteroides community never returned to its original composition during the study period, as assessed using the molecular fingerprinting technique, terminal restriction fragment length polymorphism.

POSSIBLE ROLE OF INTESTINAL MICROBIOTA IN NECROTIZING ENTEROCOLITIS (NEC)

Necrotizing enterocolitis (NEC) is among the most severe conditions that can affect premature infants and is a life-threatening medical and surgical emergency. It remains an enigmatic and potentially devastating condition with high morbidity and mortality in preterm infants.37 Although the etiology of NEC remains unknown, initial bacterial colonization could play a pivotal role in the development of NEC.

To further explore the putative relationship between pathogenic microorganisms and NEC, a prospective case control study in 12 preterm infants examined early intestinal bacterial colonization and NEC and the putative role of Clostridium. PCR and temporal TGGE of 16S ribosomal DNA were used to detect the establishment of bacterial communities in the digestive tract.38 The group included three infants with NEC and nine control infants without evidence of NEC who were matched for gestational age and birth weight. Stool samples were collected at weekly intervals from all infants. A significant temporal relationship was established between early colonization by Clostridium and the later development of NEC. A salient feature of the bacteriological pattern was observed only in the three infants who later developed NEC: A band corresponding to the Clostridium perfringens subgroup could be detected in early samples, before the NEC diagnosis. There was no evidence of this specific band in any of the nine controls.

Using 16S rRNA gene PCR, the bowel flora of preterm infants with and without NEC was evaluated.39 Fecal samples from 32 preterm infants, including samples from 10 infants with NEC, were examined by culture and PCR amplification of the 16S rRNA gene. In this study, uncultured bacteria detected by PCR-DGGE were no more frequent in the fecal samples of infants with NEC than in the samples of infants without NEC; however, these findings do not exclude the possibility of unrecognized bacteria being associated with the mucosa of the small intestine of infants with NEC. More extensive studies employing sequence analysis are indicated.

CONCLUSION

The importance of gut microbiota for human health has been increasingly recognized, but the early bacterial colonization of the GI tract in premature babies is not yet completely understood. The mechanisms underlying these interactions are complex and are influenced by many factors. Improved knowledge of the normal evolution of the intestinal microbiota of premature babies, the impact of antibiotics, human milk, or premature formula, as well as several other perinatal and postnatal perturbations on this evolution will provide essential information for improving our understanding of how the development of this important bacterial community relates to disease and/or subsequent health.

REFERENCES

View Abstract