Bacteria can be applied as biofertilizers to improve crop growth in phosphorus (P)-limited conditions. However, their mode of action in a soil environment is still elusive. We used the strain ALC_02 as a case study to elucidate how Bacillus subtilis affects dwarf tomato cultivated in soil-filled rhizoboxes over time. ALC_02 improved plant P acquisition by increasing the size and P content of P-limited plants. We assessed three possible mechanisms, namely root growth stimulation, root hair elongation, and solubilization of soil P. ALC_02 produced auxin, and inoculation with ALC_02 promoted root growth. ALC_02 promoted root hair elongation as the earliest observed response and colonized root hairs specifically. Root and root hair growth stimulation was associated with a subsequent increase in plant P content, indicating that a better soil exploration by the root system improved plant P acquisition. Furthermore, ALC_02 affected the plant-available P content in sterilized soil differently over time and released P from native P pools in the soil. Collectively, ALC_02 exhibited all three mechanisms in a soil environment. To our knowledge, bacterial P biofertilizers have not been reported to colonize and elongate root hairs in the soil so far, and we propose that these traits contribute to the overall effect of ALC_02. The knowledge gained in this research can be applied in the future quest for bacterial P biofertilizers, where we recommend assessing all three parameters, not only root growth and P solubilization, but also root hair elongation. This will ultimately support the development of sustainable agricultural practices.

Plant growth-promoting microbes (PGPM) can enhance crop yield and health, but knowledge of their mode-of-action is limited. We studied the influence of two Bacillus subtilis strains, the natural isolate ALC_02 and the domesticated 168 Gö, on Arabidopsis and hypothesized that they modify the root architecture by modulating hormone transport or signaling. Both bacteria promoted increase of shoot and root surface area in vitro, but through different root anatomical traits. Mutant plants deficient in auxin transport or signaling responded less to the bacterial strains than the wild-type, and application of the auxin transport inhibitor NPA strongly reduced the influence of the strains. Both bacteria produced auxin and enhanced shoot auxin levels in DR5::GUS reporter plants. Accordingly, most of the beneficial effects of the strains were dependent on functional auxin transport and signaling, while only 168 Gö depended on functional ethylene signaling. As expected, only ALC_02 stimulated plant growth in soil, unlike 168 Gö that was previously reported to have reduced biofilms. Collectively, the results highlight that B. subtilis strains can have strikingly different plant growth-promoting properties, dependent on what experimental setup they are tested in, and the importance of choosing the right PGPM for a desired root phenotype.

Spore-forming, plant growth-promoting bacteria (PGPR) offer extraordinary opportunities for increasing plant productivity in climate change scenarios. Plant-water relationships, root development and photosynthetic performances are all key aspects of plant physiology determining yield, and the ability of PGPR to influence these in a coordinated manner is crucial for their success. In this study, we dissected the mode of action of a commercial Bacillus paralicheniformis FMCH001 in promoting soybean (Glycine max, seed variety: Sculptor) establishment in well-watered and drought conditions. We found that FMCH001 colonizes the roots, improved root growth and allowed plants to absorb more nutrients from the soil. FMCH001 inoculation had no effect on abscisic acid in leaf or xylem sap, while significantly improved photosynthesis rate, stomatal conductance and transpiration rate at 28 days after planting when drought stress exposed for 7 days, with depressed leaf water potential and osmotic potential. Moreover, the bacterium increased water use efficiency and the inoculated soybean plants exposed to drought used 22.94% less water as compared to control, despite producing comparable biomass. We propose that the ability of the bacterium to promote root growth and also modulate plant water relations are key mechanisms that allow FMCH001 to promote growth and survival in dicotyledon plants.

Root-knot nematodes (RKNs, Meloidogyne spp.) are obligate plant parasites that constitute a significant pest for agriculture worldwide. They penetrate the plant roots, reducing the uptake of water and nutrients, causing a significant impact on crop yield. One alternative on focus now for nematode management is biological control. Rhizobacteria within the Bacillus genus show multiple modes of action against plant-parasitic nematodes (PPNs) that can act alone or in combination. In this context, we evaluated a dual-strain bacteria combination ( B. paralicheniformi FMCH001 and B. subtilis FMCH002) to reduce nematode infection in tomato plants. We evaluated mortality of larvae from Meloidogyne javanica in vitro , as well as eggs hatching after the treatment. Atraction, penetration, establishment, and reproduction assays in vitro or in pots in tomato plants infected with M. javanica and treated/ untreated with the dual-strain bacteria combination were also performed. Additionally, morphometric parameters comparing giant cells size from galls of treated and untreated plants by using confocal microscopy were also measured. The results showed that this combination of strains has nematicidal properties in the pre-infection phase by decreasing the egg-hatching, juvenile survival, and attractiveness to the roots. Furthermore, nematode establishment, gall formation, and, remarkably, giant cell development was severely impaired after the bacterial treatment, suggesting interference with morphogenetic mechanisms induced by the nematode during GCs development within the plant. Nematode reproduction in tomato plants was reduced independently of the application mode in soil, before or after bacterial treatment. The dual-strain combination was also effective against other PPNs (i.e. Pratylenchus spp.) and in different crops (soybean). Therefore, combining B. paralicheniformis FMCH001 and B. subtilis FMCH002 is an efficient agent for the biological control of Meloidogyne spp. by interfering with different stages of the nematode cycle as a result of multiple modes of action.

Increasing agricultural losses due to biotic and abiotic stresses caused by climate change challenge food security worldwide. A promising strategy to sustain crop productivity under conditions of limited water availability is the use of plant growth promoting rhizobacteria (PGPR). Here, the effects of spore forming Bacillus licheniformis (FMCH001) on growth and physiology of maize (Zea mays L. cv. Ronaldinho) under well-watered and drought stressed conditions were investigated. Pot experiments were conducted in the automated high-throughput phenotyping platform PhenoLab and under greenhouse conditions. Results of the PhenoLab experiments showed that plants inoculated with B. licheniformis FMCH001 exhibited increased root dry weight (DW) and plant water use efficiency (WUE) compared to uninoculated plants. In greenhouse experiments, root and shoot DW significantly increased by more than 15% in inoculated plants compared to uninoculated control plants. Also, the WUE increased in FMCH001 plants up to 46% in both well-watered and drought stressed plants. Root and shoot activities of 11 carbohydrate and eight antioxidative enzymes were characterized in response to FMCH001 treatments. This showed a higher antioxidant activity of catalase (CAT) in roots of FMCH001 treated plants compared to uninoculated plants. The higher CAT activity was observed irrespective of the water regime. These findings show that seed coating with Gram positive spore forming B. licheniformis could be used as biostimulants for enhancing plant WUE under both normal and drought stress conditions.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. The microbiota of pigs influenced by diet texture and severity of infection Lars Mølbak, Kaare Johnsen, Mette Boye, Tim K. Jensen, Markku Johansen, Kristian Møller, Thomas D. Leser

Terminal-restriction fragment length polymorphism (TRFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at –20°C or –80°C. In this study, samples of intestines from five different pigs were stored at –80°C and –20°C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at –80°C compared to the samples stored at –20°C. Thus it is recommended from this study that samples of intestinal content are stored at –20°C before use for bacterial community analysis, instead of the current practice at –80 C.

BACKGROUND:Necrotizing enterocolitis (NEC) is associated with changes in the luminal gut microbiota. It is not known whether the mucosa-associated microbiota is affected by NEC and stimulates inflammatory lesions.OBJECTIVE:We hypothesized that the density of the mucosa-associated microbiota correlates with NEC severity in preterm pigs and that in vitro infection with increasing densities of Clostridium perfringens, which has been associated with NEC in preterm infants, would lead to a transcriptional response related to the inflammatory conditions of NEC.METHODS:First, we determined the density of total bacteria and C. perfringens in the distal small intestinal mucosa of 58 NEC and healthy preterm pigs using quantitative PCR. Next, we analyzed in IPEC-J2 cells the effect of different infection densities of C. perfringens type A on the expression of genes related to intestinal function and immune response.RESULTS:Total bacterial and C. perfringens densities were higher in NEC versus healthy pigs and correlated positively with NEC severity. In IPEC-J2 cells expression levels of inflammation-related genes (CCL5, NFKBIA, IL8, IL1RN, and TNFAIP3) increased, while the expression of the sodium/glucose co-transporter (SLC5A1) decreased, with increasing density of C. perfringens.CONCLUSIONS:Total bacterial and C. perfringens densities were higher in NEC versus healthy pigs and correlated positively with NEC severity. In IPEC-J2 cells expression levels of inflammation-related genes (CCL5, NFKBIA, IL8, IL1RN, and TNFAIP3) increased, while the expression of the sodium/glucose co-transporter (SLC5A1) decreased, with increasing density of C. perfringens.

Background In recent years, new neonatal porcine diarrhoea (NNPD) of unknown aetiology has emerged in Denmark. NNPD affects piglets during the first week of life and results in impaired welfare, decreased weight gain, and in the worst-case scenario death. Commonly used preventative interventions such as vaccination or treatment with antibiotics, have a limited effect on NNPD. Previous studies have investigated the clinical manifestations, histopathology, and to some extent, microbiological findings; however, these studies were either inconclusive or suggested that Enterococci, possibly in interaction with Escherichia coli , contribute to the aetiology of NNPD. This study examined ileal and colonic luminal contents of 50 control piglets and 52 NNPD piglets by means of the qPCR-based Gut Microbiotassay and 16 samples by 454 sequencing to study the composition of the bacterial gut microbiota in relation to NNPD. Results NNPD was associated with a diminished quantity of bacteria from the phyla Actinobacteria and Firmicutes while genus Enterococcus was more than 24 times more abundant in diarrhoeic piglets. The number of bacteria from the phylum Fusobacteria was also doubled in piglets suffering from diarrhoea. With increasing age, the gut microbiota of NNPD affected piglet and control piglets became more diverse. Independent of diarrhoeic status, piglets from first parity sows (gilts) possessed significantly more bacteria from family Enterobacteriaceae and species E. coli , and fewer bacteria from phylum Firmicutes. Piglets born to gilts had 25 times higher odds of having NNPD compared with piglets born to multiparous sows. Finally, the co-occurrence of genus Enterococcus and species E. coli contributed to the risk of having NNPD. Conclusion The results of this study support previous findings that points towards genus Enterococcus and species E. coli to be involved in the pathogenesis of NNPD. Moreover, the results indicate that NNPD is associated with a disturbed bacterial composition and larger variation between the diarrhoeic piglets.

Compared with lean humans, the gut microbiota is altered in the obese. Whether these changes are due to an obesogenic diet, and whether the microbiota contributes to adiposity is currently discussed. In the cat population, where obesity is also prevalent, gut microbiome changes associated with obesity have not been studied. Consequently, the aim of this study was to compare the gut microbiota of lean cats, with that of overweight and obese cats. Seventy-seven rescue-shelter cats housed for ≥3 consecutive days were included in the study. Faecal samples were obtained by rectal swab and, when available, by a paired litter box sample. Body condition was assessed using a 9-point scoring system. DNA was extracted, and the 16S rRNA gene was amplified with a high-throughput quantitative real-time PCR chip. Overweight and obese cats had a significantly different gut microbiota compared to lean cats (p < 0.05), but this finding could not be linked to differences in specific bacterial groups. The rectal samples obtained higher DNA concentration than litter box samples (p < 0.0001). In conclusion, overweight and obese cats seem to have an altered gut microbiome as compared to lean cats.

Background & aims: : The aim of this study was to investigate the spectrum of colonic microflora in patients with colitis and if this could be altered with one month's treatment with synbiotics.Methods: This was a pilot study in which patients were randomised to either receive a synbiotics preparation for a month and then "crossed over" to receive a placebo, or alternatively to receive the placebo first followed in the second month by synbiotic. Stool samples were collected on entry into the study and then at the end of first and second months respectively. Colonic microflora was measured by terminal restriction fragment length polymorphism technique. Quantitative PCR was used to determine the concentration of individual species.Results: Sixteen patients completed the study of whom 8 had Crohn's colitis and 8 had ulcerative colitis. Their median age was 62 (IQR 50-65) years. An average of 22 terminal restriction fragments (T-RF's) was identified in each patient. Dice cluster analysis showed that each patient had a unique microbial composition which did not change significantly at different time points in the study, irrespective of whether they had probiotics or the placebo. Probiotic organisms were identified in stool samples but did not alter overall spectrum of microflora. In this pilot study we were unable to identify any specific characteristics related to nature of colitis.Conclusions: This study suggests that there is no difference in colonic microflora between patients with Crohn's or Ulcerative colitis and that the spectrum of bacteria was not altered by synbiotic administration. (C) 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

Preterm birth, bacterial colonization, and formula feeding predispose to necrotizing enterocolitis (NEC). Antibiotics are commonly administered to prevent sepsis in preterm infants, but it is not known whether this affects intestinal immunity and NEC resistance. We hypothesized that broad-spectrum antibiotic treatment improves NEC resistance and intestinal structure, function, and immunity in neonates. Caesarean-delivered preterm pigs were fed 3 days of parenteral nutrition followed by 2 days of enteral formula. Immediately after birth, they were assigned to receive either antibiotics (oral and parenteral doses of gentamycin, ampicillin, and metronidazole, ANTI, n = 11) or saline in the control group (CON, n = 13), given twice daily. NEC lesions and intestinal structure, function, microbiology, and immunity markers were recorded. None of the ANTI but 85% of the CON pigs developed NEC lesions by day 5 (0/11 vs. 11/13, P < 0.05). ANTI pigs had higher intestinal villi (+60%), digestive enzyme activities (+53-73%), and goblet cell densities (+110%) and lower myeloperoxidase (-51%) and colonic microbial density (10(5) vs. 10(10) colony-forming units, all P < 0.05). Microarray transcriptomics showed strong downregulation of genes related to inflammation and innate immune response to microbiota and marked upregulation of genes related to amino acid metabolism, in particular threonine, glucose transport systems, and cell cycle in 5-day-old ANTI pigs. In a follow-up experiment, 5 days of antibiotics prevented NEC at least until day 10. Neonatal prophylactic antibiotics effectively reduced gut bacterial load, prevented NEC, intestinal atrophy, dysfunction, and inflammation and enhanced expression of genes related to gut metabolism and immunity in preterm pigs.

You might find this additional info useful...This article cites€53 articles, 27 of which you can access for free at: http://ajpgi.physiology.org/content/304/3/G300.full#ref-list-1Updated information and services including high resolution figures, can be found at: http://ajpgi.physiology.org/content/304/3/G300.fullAdditional material and information about AJP - Gastrointestinal and Liver Physiology can be found at:http://www.the-aps.org/publications/ajpgiThis information is current as of June 18, 2013.

Preterm neonates are susceptible to gastrointestinal disorders such as necrotizing enterocolitis (NEC). Maternal milk and colostrum protects against NEC via growth promoting, immunomodulatory, and antimicrobial factors. The fetal enteral diet amniotic fluid (AF), contains similar components, and we hypothesized that postnatal AF administration reduces inflammatory responses and NEC in preterm neonates. Preterm pigs (92% gestation) were delivered by caesarean section and fed parental nutrition (2 days) followed by enteral (2 days) porcine colostrum (COLOS, n = 7), infant formula (FORM, n = 13), or AF supplied before and after introduction of formula (AF, n = 10) in experiment 1, and supplied only during the enteral feeding period in experiment 2 (FORM, n = 16; AF, n = 14). The NEC score was reduced in both AF and COLOS pigs, relative to FORM, when AF was provided prior to full enteral feeding (9.9 and 7.7 compared with 17.3, P < 0.05). There was no effect of AF when provided only during enteral feeding. AF pigs showed decreased bacterial abundance in colon and intestinal inflammation-related genes (e.g., TNF-α, IL-1α, IL-6, NOS) were downregulated, relative to FORM pigs with NEC. Anti-inflammatory properties of AF were supported by delayed maturation and decreased TNF-α production in murine dendritic cells, as well as increased proliferation and migration, and downregulation of IL-6 expression in intestinal cells (IEC-6, IPEC-J2). Like colostrum, AF may reduce NEC development in preterm neonates by suppressing the proinflammatory responses to enteral formula feeding and gut colonization when provided before the onset of NEC.

This study aimed to assess whether inclusion of chicory or lupine (prebiotics) in the diet of pre-slaughter pigs for just 1 or 2 weeks could change the composition of their intestinal microbiota, stimulate the growth of bifidobacteria and help to lower the amount of thermoplilic Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli), which are a major cause of food-borne infections in humans. A total of 48 pigs that had an initial live weight of 90 kg were fed with either a lupine (organic concentrate with 25% blue lupine seeds), chicory (organic concentrate with 10% dried chicory roots) or control (100% organic concentrate) diet for 1 week (24 pigs) or 2 weeks (24 pigs) before slaughter. The Campylobacter spp. level in rectal faecal samples after 0, 1 and 2 weeks of feeding and in the luminal content from ileum, caecum and colon at slaughter was determined by direct plating on modified charcoal-cefoperazone-deoxycholate agar plates. DNA extracted from the luminal content of distal ileum and caecum was used for terminal restriction fragment length polymorphism (T-RFLP) analysis of the composition of intestinal microbiota and for measuring the amount of bifidobacterial and total bacterial DNA by quantitative real-time PCR (qPCR). Campylobacter spp. were excreted by all pigs and present in the luminal content from distal ileum to midway colon with particularly high numbers in the caecum, but the excretion was reduced by 10-fold in pigs fed lupines for 1 week as compared with control- and chicory-fed pigs (mean log10 2.9 v. 4.1 CFU/g; P < 0.05). The qPCR analysis showed that feeding with lupines resulted in higher levels of bifidobacteria in caecum as compared with the other diets (P < 0.05). T-RFLP analysis showed that four of the most abundant bacteria with terminal restriction fragment values >5% relative to the intensity of total abundance differed between the feed treatments (P < 0.05). Therefore, this study showed that even a short-term alternative feeding strategy with prebiotics in the diet of pre-slaughter pigs elicited changes in the composition of the intestinal microbiota, where lupine increased the level of bifidobacteria in caecum and reduced the Campylobacter spp. excretion level after 1 week.

The overall aim of this study was to describe uterine bacterial flora during the postpartum period in Danish Holstein cows using the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method. This method produces a pattern of nucleic acid fragments from the microorganisms present, reflecting the “fingerprint” of the actual microbial flora. As well as characterizing changes in flora with time from calving and between herds, data were examined for strong relations between uterine bacterial flora, calving management and uterine condition. In total 125 Holstein cows from five herds were included, and for each cow calving management was recorded. Cows were clinically examined on average 8 (range 0–19) and 28 (range 22–38) days after calving, and a uterine sample was taken for bacterial identification using T-RFLP. Milk samples were taken weekly for progesterone analysis. Bacteria were found in all cows at both examinations, and the flora was composed of many species, including species not traditionally reported to be present in the bovine uterus. The bacterial composition differed according to days from calving and herd. In all five herds Fusobacterium necrophorum, Pseudomonas/Acinetobacter and Bacteroides/Sphingobacterium/Prevotellaceae were among the most common at both examinations. In four herds there was a percentage decrease of F. necrophorum from first to second examination, and in all herds there was a percentage increase of Pseudomonas/Acinetobacter from first to second examination. No differences in bacterial flora were found between cows with different uterine scores, which were influenced by herd, calving difficulty and retained placenta.

Background Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). Results A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P <0.0001) and in non-cloned control pigs (r=0.9, P <0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P <0.02) and non cloned-control pigs (r=0.45; P <0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=−0.33, P <0.04), but not in the non-cloned control pigs. Conclusion The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.

Preterm infants are susceptible to necrotizing enterocolitis (NEC). Using preterm pigs, we determined whether a whey-casein-based formula would be superior to a formula based on whey protein alone. Twenty cesarean-derived preterm pigs (92% gestation) were given total parenteral nutrition for 36 h followed by 30 h of enteral feeding with whey [protein fraction of milk formula based on whey (WHEY); n = 11] or casein and/or whey [protein fraction of milk formula based on a combination of casein and whey (CASEIN); n = 9]-based formulas. Sugar absorptive function was investigated at 6 and 30 h after initiation of enteral feeding using bolus feedings with galactose and mannitol. Pigs were killed after the last in vivo sugar absorption test and evaluated for NEC and the mid intestine was used for ex vivo measurements of hexose absorption. Microbiota profile and short chain fatty acid (SCFA) levels were studied in gut contents. Severity of NEC lesions was similar between diet groups but galactose absorption was markedly higher in CASEIN than in WHEY (P < 0.01) although only 6 h after the start of the enteral feeding period. There were no differences in ex vivo (14)C-D-glucose uptake, digestive enzymes, microbiota profile, or SCFA concentration. Casein may transiently stimulate intestinal sugar absorption but has limited effects on gut structure, microbiota, and NEC in preterm pigs.