What Is Known
- Preterm birth is associated with disturbed gut microbial colonization.
- Gut microbial colonization of infants is influenced by delivery mode, diet, environmental exposure and maturity at birth, but the effect of the individual factors remain poorly understood.
What Is New
- Using preterm piglets as model for preterm infants, it is shown that preterm birth has effects on gut microbiota composition and metabolism in the first 4 weeks of life.
- Conversely, in piglets delayed introduction of enteral feeding after preterm birth has only a temporary effect on the gut microbiota composition and metabolism.
Infants born very preterm are born with an immature gastrointestinal tract (GIT) (1). Due to their immature GIT, feeding intolerance is common among preterm infants and most preterm infants receive partial or total parenteral nutrition (PN) the first weeks of life (1–3). Relying entirely on total PN (TPN) for a prolonged period after birth is associated with mucosal atrophy (4), cholestasis, and other complications, such as catheter-related sepsis (5,6). Different feeding regimes exist but at many hospitals, enteral nutrition is often introduced gradually (minimal enteral nutrition [MEN]) in combination with PN during the first weeks of life with some observational studies indicating that this approach protects against development of necrotizing enterocolitis (NEC) (3). A recent Cochrane review (7) and a randomized clinical trial (8), however, showed that slow advancement of enteral feed volumes does not reduce the risk of NEC or risk of death and recommend more rapid progression towards full enteral feeding, though for extremely low birthweight infants (<750 g) there are some indications that slow introduction of enteral feeding might be beneficial with respect to reduced NEC incidence (9). Mothers’ milk is the optimal diet to support gut immunity, digestive functions, and growth (10) but lactation is often delayed after preterm birth, requiring the use of less optimal alternatives, such as human donor milk (if available) or infant formula (3).
Shortly after birth, the near-sterile infant GIT is colonized by a wide range of microorganisms (11,12). The progression of intestinal gut microbiota (GM) colonization is influenced by a range of factors, such as mode of delivery, maternal microbiota, gestational age (GA), genetics, mode of feeding, and exposure to antibiotics (13–15). During the first days after birth, the human infant gut is colonized by facultative anaerobes such as Enterobacteriaceae, Streptococcus, Enterococcus and Staphylococcus, but gradually the GM becomes dominated by bifidobacteria and obligate anaerobic bacteria, such as eubacteria and clostridia (13,14,16). In preterm infants, GM diversity has been shown to be lower compared with term infants of the same postnatal age (17), with the preterm GM showing temporal instability and high inter-individual variability (18,19). In a longitudinal study, La Rosa et al (20) demonstrated that during the first 5–6 weeks of life the preterm infant GM was dominated by taxa belonging to classes Bacilli, Clostridia and Gammaproteobacteria, and the colonization pattern in the preterm infants was determined by postconceptional age rather than postnatal age. However, interpretations relative to term infants are difficult because the colonization pattern in preterm infants is influenced by clinical care procedures at hospitals, including use of antibiotics. A postnatal comparison of gut colonization in caesarean-delivered preterm and term human infants, using identical dietary and clinical treatments, are thus difficult to achieve, but using preterm piglets as model for preterm infants may help to shed light on whether immaturity at birth and host-specific factors induce a different colonization pattern in preterm versus term infants.
In terms of immunity and GIT development, preterm piglets in many ways resemble preterm infants and they have a high sensitivity to NEC and sepsis (21,22). We have previously shown in preterm piglets that PN supplemented with gradually increasing volumes of bovine colostrum (EEF) was superior to EEF based on infant formula (23,24) in protecting against NEC and that EEF stimulates GIT maturation relative to TPN (24–27). Further, it has been found, that early life gradual feeding with bovine colostrum (EEF) leads to an altered GM colonization pattern relative to TPN and infant formula-based EEF-fed preterm piglets. In all cases, the GM of the preterm piglets were dominated by Proteobacteria at day 5, but colostrum-fed piglets harbored a higher proportion of clostridia resulting in increased colonic butyrate concentrations. The formula-fed pigs on the other hand harbored high proportions of Enterococcus spp. resulting in increased colonic lactate production (24). It is not known how differences in early feeding affect the GM more long-term for preterm individuals.
We hypothesized that reduced GA at birth (vs birth at term) and the first enteral feeding (vs no enteral feeding) induce changes in gut colonization that last beyond the first weeks after birth. Consequently, the aim of the present study was to investigate the influence of 2 clinically relevant feeding regimens (ie, TPN vs EEF for the first 5 days) on GM composition and function at 5 and 26 days after birth in caesarean-delivered preterm and term piglets given the same rearing conditions.
Animals, Diets and Sample Collection
The overall study design is outlined in Figure 1. All animal procedures were approved by the Danish Committee on Animal Experimentation (protocol 2012-15-2934-00193). One hundred and sixteen piglets (Danish Landrace × Large White × Duroc) were delivered by caesarean section either preterm (PT, 90% of full GA, 106 days, n = 72) or term (T, 100% gestation age, 118 days, n = 44) following previously described procedures (21,25,28). All piglets were reared individually in a piglet neonatal intensive care unit with controlled temperature, and oxygen-regulated incubators, as previously described (25,28). In brief, the piglets were block-randomized based on birth weight to receive either TPN (PT-TPN, n = 33; T-TPN, n = 20) or PN supplemented with gradually increasing enteral feeding with bovine colostrum (PT-EEF, n = 39; T-EEF, n = 24) for 5 days (25,28). For the TPN group, PN (a modified combination of Kabiven, Vitalipid, Soluvit, and Vamin, Fresenius Kabi, Bad Homburg, Germany) (21) was given at 96 mL · kg−1 · day−1 on day 1, gradually increasing to 144 mL · kg−1 · day−1 on day 5, whereas for the EEF group, enteral nutrition with bovine colostrum (Biofiber Damino, Vejen, Denmark) started at 16 mL · kg−1 · day−1 on day 1, increasing to 64 mL · kg−1 · day−1 on day 5, and this was accompanied by a reduction in PN, such that the 2 dietary regimens provided similar fluid volumes and were iso-energetic (increasing from 74 to 110 kcal · kg−1 · day−1 over the first 5 days), see (28) for further details. Importantly term and preterm pigs received identical treatments with antibiotics. To prevent diarrhoea caused by coccidia, a common porcine parasite, piglets received prophylactic toltrazuril by mouth on day 5 (20 mg/kg; Baycox, Bayer Animal Health, Leverkusen, Germany), and amoxicillin trihydrate (Paracillin Vet 70%, MSD, Animal Health, Ballerup, Denmark) was administered prophylactically in the feed (20 mg/L) on days 5 to 15, to prevent gut bacterial overgrowth, infection, and sepsis. In individual cases of diarrhea, pigs were supplied with an electrolyte mixture (Revolyt, GK Pharma, Køge, Denmark), and severe diarrhoea with suspected infectious etiology was treated with gentamicin (5 mg/piglet orally, gentamycin, ScanVet Animal Health, Fredensborg, Denmark) and enrofloxacin (5 mg/kg im; Baytril, Bayer Animal Health) for 3 consecutive days.
After day 5, all piglets were fed full enteral nutrition until day 26. First with increasing amounts of raw bovine milk (64–150 mL · kg−1 · day−1, providing 37–70 kcal · kg−1 · day−1) until day 10, and then transferred to reconstituted whole milk powder (WMP; 150–200 mL · kg−1 · day−1, Arla Foods, Viby J, Denmark) until day 26. The pigs were euthanized and sampled at day 5 or 26. Colon content was collected and stored at −80 °C until further analysis. See Hansen et al (25) for detailed procedures of sample collection.
Approximately 200 mg of colon content was weighted into a PowerSoil Bead Tube and total DNA extracted using the MoBio PowerSoil DNA isolation kit following the manufacturer's standard protocol but supplemented with an initial bead beating step (FastPrep-24 Homogeniser, 3 cycles of 15 seconds, speed 6.5). DNA concentration was measured using Nanodrop (ND-100, NanoDrop Technologies, Wilmington, DE). Extracted DNA was stored at −60 oC until further analysis.
Absolute Bacterial Quantification
Absolute quantification of bacteria in the colon content was conducted using qPCR (7500 Fast Real-time PCR system, Applied Biosystem, Foster City, CA) as described before (22). Escherichia coli K12 containing 7 rRNA gene operons (29) was used as a positive control and standard for absolute quantification.
16S rRNA Gene Amplicon Sequencing
The colon prokaryotic microbiota composition was determined using tag-encoded 16S rRNA gene MiSeq-based (Illumina, San Diego, CA) high throughput sequencing. The V3-V4 region of the 16S rRNA gene was amplified using primers compatible with the Nextera Index Kit (Illumina, adapters in bold): NXt_341_F: 5’-TCGTCGGCA GCGTCAGATG TGTATAAGAG ACAGCCTAYG GGRBGCASCAG-3’ and NXt_806_R: 5’-GTCTCGTGGG CTCGGAGATG TGTAAAGAGA CAGGGACTAC NNGGGTATC TAAT-3’. Sequencing libraries were prepared as previously described (30) and sequenced (V2 kit: paired-end 2 × 250 bp) by the National High-throughput DNA Sequencing Centre, University of Copenhagen, Denmark.
Amplicon Sequencing Data Analysis
The raw dataset containing pair-ended reads was merged, trimmed, filtered from chimeric reads, and subjected to OTU clustering using the UPARSE pipeline (31). The green genes (13.8) 16S rRNA gene collection was used as a reference database. Quantitative Insight Into Microbial Ecology (QIIME) open source software package (version 1.8.0) was used for subsequent analysis steps (32,33).
Alpha diversity expressed as observed species and Shannon indexes were computed for rarefied OTU tables (8000 reads per sample) using the alpha rarefaction workflow. Alpha diversity differences were tested by t test using the nonparametric (Monte Carlo) method (999 permutations) that is implemented in the compare alpha diversity workflow. Differences in relative abundance were analyzed using ANOVA (with False Discovery Rate [FDR] adjusted P values below 0.05 considered significant, FDR-adjusted P values are referred to as q values in the following). Principal Coordinate Analysis plots based on 10 weighted and unweighted UniFrac distances were calculated using subsampled OTU-tables (8000 reads/sample). Differences in ordination between categories were verified with Analysis of Similarities (ANOSIM) based on randomly selected distance matrices.
Short-chain Fatty Acids
The concentration of short-chain fatty acids (SCFA); acetic, propionic, butyric, isobutyric, valeric and isovaleric acid, and lactic acid in colon samples obtained at day 26 was quantified by GC-MS as described previously (34).
SCFA results and total bacterial load as determined by qPCR were analyzed by GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA). Differences between the different feeding regimes and GA (PT-EEF, PT-TPN, T-EEF, T-TPN) were analyzed by one-way ANOVA with Dunnett post-test. Pearson product-moment correlation coefficient was used to assess associations between SCFA levels and bacterial relative abundance at genera level (q < 0.05). False discovery rate (FDR)-corrected P-values < 0.05 was considered significant.
Seventy-two preterm and 44 term piglets were included in the study and assigned to different dietary regimes, as lined out in Figure 1.
Bacterial Load in Colon Content on Days 5 and 26
Total bacterial (prokaryotic) abundance expressed as 16S rRNA gene copies/g was determined by qPCR. No significant differences in bacterial total count between T and PT piglets were observed on day 5 or day 26. Similarly, the different diet regimens (EEF vs TPN) during the first 5 days of life did not influence prokaryotic absolute abundance, neither short-term (5 days) nor more long-term (26 days) (Suppl. Fig. 1, Supplemental Digital Content 1, http://links.lww.com/MPG/B561).
Gut Microbiota Composition on Days 5 and 26
Alpha diversity indices were assessed by Observed species and Shannon diversity indices (Suppl. Fig. 2A and 2B, Supplemental Digital Content 1, http://links.lww.com/MPG/B561). For term piglets, no significant differences in Observed species and Shannon diversity were observed between diets nor sampling days (day 5 vs day 26) (Suppl. Fig. 2B, Supplemental Digital Content 1, http://links.lww.com/MPG/B561). At day 5, preterm piglets tended to have reduced number of observed species and Shannon diversity index compared with term piglets, whereas from days 5 to 26, both indexes increased 2- to 3-fold (P < 0.05) independent of dietary regime (Suppl. 2A and 2B, Supplemental Digital Content 1, http://links.lww.com/MPG/B561). We observed no significant differences between piglets born preterm and term on day 26 in neither observed species nor Shannon diversity index (Suppl. Fig. 2A and 2B, Supplemental Digital Content 1, http://links.lww.com/MPG/B561).
Gut microbiota compositional analysis based on weighted and unweighted UniFrac distance metrics showed clear differences because of GA (Table 1 and Fig. 2A) at day 5. Early life diet (TPN vs MEN) also significantly influenced piglet GM short-term, but only based on unweighted UniFrac distance metrics (Table 1 and Fig. 2B). This indicates that the GM differences driven by the diet were mainly because of the low abundant taxa, while the GA influenced both high and low abundant bacterial groups.
Between day 5, where all piglets started to receive full enteral feeding with bovine milk, and day 26 the GM composition of the piglets converged and the diet effects observed on day 5 did not persist at day 26. In contrast, the observed difference between gestation age (preterm vs term piglets) on day 5 persisted until day 26 (Table 1 and Fig. 2C).
At day 5, the majority of the OTUs in all 4 groups could be classified to the Proteobacteria and Firmicutes phyla constituting an average of 62% to 94% and 5% to 28% of the prokaryotic colon community, respectively (Fig. 3). An OTU closely related to Escherichia coli (Enterobacteriaceae family) dominated the colon microbiota of all 4 groups (PT-EEF [average relative abundance 70%], PT-TPN [88%], T-EEF [61%], and T-TPN [60%]). The phylum Firmicutes was mainly represented by order Clostriadales constituting 5% to 14% in preterm piglets and 19% to 26% in the term piglets (Fig. 3).
Several taxa were found to differ between PT and T piglets fed the same diet until day 5, although after FDR correction only Erwina spp. (PT-EEF 5.3%, T-EEF 0.5%, q = 0.06) remained near significance. Similarly, several taxa differed in relative abundance between piglets of same GA fed either EEF or TPN at day 5 (Fig. 3), but again because of large inter-individual differences between the piglets, none of these remained significant after FDR correction.
At day 26, the OTU closely related to Escherichia coli had the highest abundance in the colon of most piglets, PT-EEF (45.6%), PT-TPN (44.4%), T-EEF (57.5%), T-TPN (54.7%) (Fig. 3). Genus Lactobacillus and an OTU closely related to Clostridium bolteae (Lachnospiraceae family) were predominant (11.2% [PT-EEF] and 5.5% [PT-TPN], respectively) in most preterm piglets, whereas phylum Verrucumicrobia represented solely by the genus Akkermansia were highly abundant in both term groups; T-TPN (25.7%) and T-EEF (29.4%), but almost absent in the preterm groups; PT-TPN (0.1%) and low abundant in PT-EEF piglets (3.0%) (PT-EEF vs T-EEF, q = 0.08 and PT-TPN vs T-EEF, q = 0.001).
The relative abundance of Proteobacteria decreased between days 5 and 26 in the PT piglets (PT-EEF, 78.7% at day 5, 46.2% at day 26, q = 0.02; PT-TPN, 93.9% at day 5, 45.1% at day 26, q = 0.002). Concordantly, the relative abundance of Firmicutes increased (PT-EEF; 19.6% at day 5, 48.8% at day 26, q = 0.03; PT-TPN; 5.8% at day 5, 52.9% at day 26, q = 0.002) during the same period. The shift of phyla Proteobacteria and Firmicutes in preterm piglets is partly reflected by a decline in relative abundance of Erwinia (5.3% at day 5, 0.06% at day 26, q = 0.008) and an increasing relative abundance of Dorea spp. (0.1% at day 5, 2.8% at day 26, q = 0.03) in EEF pigs. In term piglets, the relative abundance of genus Akkermansia increased between day 5 and day 26 (T-EEF; 11% at day 5, 30% at day 26 and T-TPN; 10% at day 5, 26% at day 26) but because of the large intra-group variation (Fig. 3) and in some cases, relatively small group sizes, none of these differences remained significant after FDR-correction.
Short-chain Fatty Acid Concentrations
The formation of 7 SCFAs were quantified from colon samples at day 26. Overall, term piglets had significantly higher colon concentrations of acetate and propionate at day 26 (P < 0.01, P < 0.05), whereas preterm piglets had higher levels of formate (P < 0.05) (Suppl. Fig. 3, Supplemental Digital Content 1, http://links.lww.com/MPG/B561) compared with term piglets. Butyrate, iso-butyrate, lactate, and succinate (Suppl. Fig. 3, Supplemental Digital Content 1, http://links.lww.com/MPG/B561) did not differ between term and preterm piglets at day 26. Early-life EEF (up till day 5) has in term piglets more lasting effects, as these piglets had higher luminal acetate levels on day 26 compared with term piglets receiving TPN the first 5 days of life. For preterm piglets this effect of early-life EEF was not observed.
The relative abundance of Enterococcus spp. and Lactobacillus spp. positively correlated with colon lactic acid concentrations at day 26 in the piglets born preterm (q < 0.001, r = 0.79; q < 0.0001, r = 0.57; respectively) (Suppl. Table 1, Supplemental Digital Content 2, http://links.lww.com/MPG/B562), but not in the piglets born term (almost all term piglets having low colon concentrations of lactate). The differences in colonic SCFA concentrations between term and preterm piglets on day 26 strongly indicate that premature birth not only influences early life GM composition but also have long-term influence on the GM activity profile.
In the present study, we used term and preterm piglets as a model to study the short and more long-term effects of GA and early life diet differences. All pigs were delivered by C-section and reared, antibiotic-treated, and fed under controlled and similar conditions. Using this setup, we demonstrate that although GA has long-term influence on GM development in piglets, introduction of enteral nutrition only influences GM composition more temporarily.
Preterm birth is associated with GIT immaturity and feeding intolerance (35). Therefore, preterm infants require a period of either enteral nutrition with PN support, or TPN before enteral feeding is gradually introduced (36). Whereas introduction of enteral feeding is necessary to promote proper maturation of the preterm GIT, most preterm infants experience shorter or longer periods receiving PN and developing dietary regimens allowing early and safe introduction of enteral nutrition to preterm infants for improved gut maturation is thus of great interest. Piglets born prematurely (90% GA) resemble infants born around 70% GA in terms of gut immaturity and have been found to be a suitable and pragmatic model for studying interventions targeting preterm infants (21). Using this model, we have recently demonstrated that bovine colostrum (BC) introduced in combination with PN promotes growth, gut function, digestive function, and gut immunity in preterm piglets (24,25,27), despite that these piglets are deprived of the natural immunity normally provided by sow colostrum.
Preterm birth is associated with impaired gut maturation and function, which influences gastrointestinal motility and leads to lower gut microbial diversity in human preterm infants (17,37), a trend also observed in preterm piglets in the present study (Suppl. Fig. 2, Supplemental Digital Content 1, http://links.lww.com/MPG/B561).
Five days after birth, both preterm and term piglet GM were dominated by phyla Proteobacteria and Firmicutes, which is in agreement with previous studies on the GM of preterm infants (20,35,38–43) and preterm piglets (24). In term human infants, the early life GM is often rich in bifidobacteria and Bacteriodetes, especially Bacteroides spp., because of their ability to metabolize human milk oligosaccharides from breast milk (13,44). In preterm infants, both Bifidobacterium spp. and Bacteroides spp. have been found to be low in abundance or virtually absent in the gut (20,43), which is also observed in the preterm piglets investigated in the present study. At day 26, Proteobacteria continued to be very abundant in many piglets, though with Clostridia generally being increasingly abundant. Akkermansia spp., a mucin-degrading bacterium, was almost exclusively present in the term piglets on day 26. Akkermansia spp. are occasionally detected in human infants shortly after birth and then generally increase in abundance during the first year of life (45), but in the preterm piglets investigated in the present study, this process seems to be delayed relative to the term piglets.
The introduction of gradual enteral feeding in preterm infants has been speculated to contribute the proliferation of bacterial growth in the GIT (46) and Bjornvad et al in 2008 have reported that TPN-fed pigs harbor lower counts of clostridia compared with pigs fed enterally. However, in the present study, colon bacterial abundance as determined by qPCR was comparable between all groups (ENT and TPN) and no apparent differences in clostridial diversity were observed (Suppl. Fig. 1 and Fig. 3, Supplemental Digital Content 1, http://links.lww.com/MPG/B561).
In agreement with the GM compositional data, we observed that GA has significant influence on colonic SCFA concentrations at day 26, with acetate and propionate concentrations being significantly lower and formate levels significantly higher in piglets born prematurely, relative to piglets born at term (Suppl. Fig. 3, Supplemental Digital Content 1, http://links.lww.com/MPG/B561). Early-life EEF did not influence SCFA production on day 26 in preterm piglets, whereas in the term piglets, EEF the first 5 days of life was associated with higher acetate production on day 26 compared with term TPN-fed piglets. The observation that GA has effects on SCFA production in the longer term resembles recent findings in infants (40,47) and might have long-term consequences for both gut and whole-body metabolism.
A limitation of the study is that all piglets were administered antibiotics prophylactically from days 5 to 15. The early (day 5) piglets were euthanized and sampled before antibiotics were administered and antibiotic administration did consequently not influence these samples. However, for the pigs euthanized on day 26, one cannot completely rule out that is a carry-over effect of the antibiotic administration, but on the other hand at the time of sample collection, the pigs were well hydrated and without diarrhea and had (with a few exceptions, see Fig. 2) not been exposed to antibiotics since day 15. A further limitation is obviously the use of an animal model to resemble humans, but the used animal model resemble the GIT physiology of preterm infants in many ways (21), and thus represent an attractive model to study, for example, the influence of dietary regimes on GM development, as done in the present study.
In conclusion, we show that the establishment of GM in early life is influenced by early feeding strategy and GA at birth, but only the GA effects have a clear effect beyond the first week after birth. This influenced not only GM composition, but also GM function, as reflected in altered colonic SCFA production on day 26.
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