Neonatal prophylactic antibiotics after preterm birth affect plasma proteome and immune development in pigs.

BACKGROUND: Most preterm infants receive antibiotics to prevent serious infections shortly after birth. However, prolonged antibiotic treatment predisposes to gut dysbiosis and late-onset sepsis. Using preterm pigs as model, we hypothesized that neonatal prophylactic antibiotics impair systemic immune development beyond the days of antibiotic treatment. METHODS: Preterm pigs (90% gestation) were fed formula for 9 days, treated with sterile water (CON) or enteral antibiotics from day 1 to 4. On days 5 and 9, blood was collected for haematology, in vitro LPS stimulation, and plasma proteomics. RESULTS: Antibiotic treatment altered the abundance of 21 and 47 plasma proteins on days 5 and 9, representing 6.6% and 14.8% of the total annotated proteins, respectively. Most antibiotics-induced proteome changes related to complement cascade, neutrophil degranulation, and acute phase responses. Neutrophil and lymphocyte counts were higher in antibiotics-treated pigs on day 5 but did not change from days 5-9, in contrast to increasing cell counts in CON. The antibiotics treatment suppressed TNF-alpha and IL-10 responses to in vitro LPS challenge on day 5, 7 and 9. CONCLUSION: Few days of antibiotics treatment following preterm birth alter the plasma proteome and inhibit systemic immune development, even beyond the days of treatment. IMPACT: 1. Neonatal prophylactic antibiotics alter the plasma proteome and suppress systemic immune development in preterm pigs 2. The effects of prophylactic antibiotics last beyond the days of treatment. 3. Neonatal antibiotics treatment for compromised human newborns may predispose to longer-term risks of impaired immunity and infections.

Physical Activity and Spatial Memory Are Minimally Affected by Moderate Growth Restriction in Preterm Piglets.

BACKGROUND: Preterm birth is associated with impaired brain functions, but it is unknown whether fetal growth restriction (GR) makes these deficits worse. Using piglets as a model for preterm infants, we hypothesized that moderate GR reduces growth rate, physical activity, and spatial memory in the first weeks after preterm birth. METHODS: Preterm pigs were delivered by caesarean section and fed until 19 days (n = 830 from 55 pregnant sows) and received intensive clinical care. GR pigs were classified as animals with the lowest 5-20% percentile birth weight within each litter and were compared with litter-mate controls (21-100% percentile birth weight). Basic motor skill development, physical activity, and morbidities (e.g., necrotizing enterocolitis) were recorded within the first week. Weight of internal organs and data from a T-maze spatial memory test were noted until 19 days. RESULTS: Moderate GR and control preterm pigs (birth weights 728 ± 140 and 1,019 ± 204 g, respectively) showed similar relative weights of internal organs (relative to body), except higher adrenal gland weights in GR pigs (+20-50%, p < 0.05). This was associated with a tendency to higher plasma cortisol (p < 0.05 on day 11). GR preterm pigs showed delayed ability to stand and walk (days 2-5, p < 0.01), but physical activity and proportion of correct choices in a T-maze test (70.3 vs. 71.6%) were similar. CONCLUSION: Moderate GR has limited effect on motor function and spatial memory in the early postnatal period of preterm pigs, despite some initial delays in basic motor skills. In the postnatal period, moderately growth-restricted preterm infants may adapt well with regards to organ growth and neurodevelopment.

Prenatal Intra-Amniotic Endotoxin Induces Fetal Gut and Lung Immune Responses and Postnatal Systemic Inflammation in Preterm Pigs.

Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS-induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1ß levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses.

Common and distinct variation in data fusion of designed experimental data.

INTRODUCTION: Integrative analysis of multiple data sets can provide complementary information about the studied biological system. However, data fusion of multiple biological data sets can be complicated as data sets might contain different sources of variation due to underlying experimental factors. Therefore, taking the experimental design of data sets into account could be of importance in data fusion concept. OBJECTIVES: In the present work, we aim to incorporate the experimental design information in the integrative analysis of multiple designed data sets. METHODS: Here we describe penalized exponential ANOVA simultaneous component analysis (PE-ASCA), a new method for integrative analysis of data sets from multiple compartments or analytical platforms with the same underlying experimental design. RESULTS: Using two simulated cases, the result of simultaneous component analysis (SCA), penalized exponential simultaneous component analysis (P-ESCA) and ANOVA-simultaneous component analysis (ASCA) are compared with the proposed method. Furthermore, real metabolomics data obtained from NMR analysis of two different brains tissues (hypothalamus and midbrain) from the same piglets with an underlying experimental design is investigated by PE-ASCA. CONCLUSIONS: This method provides an improved understanding of the common and distinct variation in response to different experimental factors.

Brain Barrier Disruption and Region-Specific Neuronal Degeneration during Necrotizing Enterocolitis in Preterm Pigs.

Necrotizing enterocolitis (NEC) increases the risk of brain injury and impaired neurodevelopment. Rapid brain maturation prior to birth may explain why preterm brains are particularly vulnerable to serious infections. Using pigs as models, we hypothesized that preterm birth was associated with altered blood-cerebrospinal fluid (CSF) barrier (BCSFB) function and cerebral structural deficits, and that NEC was associated with systemic inflammation, BCSFB disruption, and neuroinflammation. First, cesarean-delivered preterm and term pigs (n = 43-44) were euthanized at birth to investigate BCSFB function and markers of brain structural maturation, or on day 5 to measure markers of blood-brain barrier maturation in the hippocampus and striatum (experiment 1). Next, preterm pigs (n = 162) were fed increasing volumes of infant formula to assess NEC lesions, systemic inflammation, BCSFB permeability, cerebral histopathology, hippocampal micro-glial density, and cytokine levels on day 5 (experiments 2 and 3). In experiment 1, preterm newborns had increased CSF-plasma ratios of albumin and raffinose, reduced CSF glucose levels, as well as increased cerebral hydration and reduced white matter myelination compared with term animals. We observed lower hippocampal (but not striatal) perivascular astrocyte coverage for the first 5 days after preterm birth, accompanied by altered cell junction protein levels. In experiments 2 and- 3, piglets with severe NEC lesions showed reduced blood thrombocytes and increased plasma C-reactive protein and interleukin-6 levels. NEC was associated with increased CSF-plasma albumin and raffinose ratios, reduced CSF leukocyte numbers, and increased cerebral hydration. In the hippocampus, NEC was associated with pyramidal neuron loss and increased interleukin-6 levels. In the short term, NEC did not affect cerebral myelination or microglia density. In conclusion, altered BCSFB properties and brain structural deficits were observed in pigs after preterm birth. Acute gastrointestinal NEC lesions were associated with systemic inflammation, increased BCSFB permeability and region-specific neuronal damage. The results demonstrate the importance of early interventions against NEC to prevent brain injury in preterm infants.

Transfer of modified gut viromes improves symptoms associated with metabolic syndrome in obese male mice.

Metabolic syndrome encompasses amongst other conditions like obesity and type-2 diabetes and is associated with gut microbiome (GM) dysbiosis. Fecal microbiota transplantation (FMT) has been explored to treat metabolic syndrome by restoring the GM; however, concerns on accidentally transferring pathogenic microbes remain. As a safer alternative, fecal virome transplantation (FVT, sterile-filtrated feces) has the advantage over FMT in that mainly bacteriophages are transferred. FVT from lean male donors have shown promise in alleviating the metabolic effects of high-fat diet in a preclinical mouse study. However, FVT still carries the risk of eukaryotic viral infections. To address this, recently developed methods are applied for removing or inactivating eukaryotic viruses in the viral component of FVT. Modified FVTs are compared with unmodified FVT and saline in a diet-induced obesity model on male C57BL/6 N mice. Contrasted with obese control, mice administered a modified FVT (nearly depleted for eukaryotic viruses) exhibits enhanced blood glucose clearance but not weight loss. The unmodified FVT improves liver pathology and reduces the proportions of immune cells in the adipose tissue with a non-uniform response. GM analysis suggests that bacteriophage-mediated GM modulation influences outcomes. Optimizing these approaches could lead to the development of safe bacteriophage-based therapies targeting metabolic syndrome through GM restoration.

Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.

BACKGROUND: Fecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinder their wider clinical application in treating less acute diseases. METHODS: To overcome these challenges, we developed methods to broaden FVT's clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: (1) chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), (2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and (3) pyronin-Y treatment to inhibit RNA virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. RESULTS: FVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels, and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. CONCLUSIONS: This proof-of-concept study shows that specific modifications of FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat propagation of donor phages emerging as promising approaches. Video Abstract.

A Gently Processed Skim Milk-Derived Whey Protein Concentrate for Infant Formula: Effects on Gut Development and Immunity in Preterm Pigs.

SCOPE: Processing of whey protein concentrate (WPC) for infant formulas may induce protein modifications with severe consequences for preterm newborn development. The study investigates how conventional WPC and a gently processed skim milk-derived WPC (SPC) affect gut and immune development after birth. METHODS AND RESULTS: Newborn, preterm pigs used as a model of preterm infants were fed formula containing WPC, SPC, extra heat-treated SPC (HT-SPC), or stored HT-SPC (HTS-SPC) for 5 days. SPC contained no protein aggregates and more native lactoferrin, and despite higher Maillard reaction product (MRP) formation, the clinical response and most gut and immune parameters are similar to WPC pigs. SPC feeding negatively impacts intestinal MRP accumulation, mucosa, and bacterial diversity. In contrast, circulating T-cells are decreased and oxidative stress- and inflammation-related genes are upregulated in WPC pigs. Protein aggregation and MRP formation increase in HTS-SPC, leading to reduced antibacterial activity, lactase/maltase ratio, circulating neutrophils, and cytotoxic T-cells besides increased gut MRP accumulation and expression of TNFAIP3. CONCLUSION: The gently processed SPC has more native protein, but higher MRP levels than WPC, resulting in similar tolerability but subclinical adverse gut effects in preterm pigs. Additional heat treatment and storage further induce MRP formation, gut inflammation, and intestinal mucosal damage.

Transplantation of fecal filtrate to neonatal pigs reduces post-weaning diarrhea: A pilot study.

Post-weaning diarrhea (PWD) remains a major source of mortality and morbidity in swine production. Transplantation of bacteria-free filtrate of feces (fecal filtrate transplant, FFT) has shown gut protective effects in neonatal pigs, and early postnatal establishment of the gut microbiome is suggested to determine later stability and robustness of the gut. We, therefore, hypothesized that early postnatal transplantation of bacteria-free feces would have a protective effect against PWD. Using fecal filtrates derived from healthy lactating sows, we compared oral administration of fecal filtrate transplantation (FFT, n = 20) and saline (CON, n = 18) in newborn piglets. We assessed growth, diarrhea prevalence, blood parameters, organ measurements, morphology, and gut brush border enzymes and analyzed luminal bacterial composition using 16S rRNA gene amplicon sequencing. The two groups showed similar average daily gain (ADG) during the suckling period, whereas in the post-weaning period, a negative ADG was observed in both groups. While diarrhea was largely absent in both groups before weaning, there was a lower diarrhea prevalence on days 27 (p = 2.07*10-9), 28 (p = 0.04), and 35 (p = 0.04) in the FFT group relative to CON. At weaning on day 27, the FFT group had higher numbers of red blood cells, monocytes, and lymphocytes, while on day 35, i.e., 1 week after weaning, the two groups were similar regarding hematology. The biochemical profile was largely similar between FFT and CON on days 27 and 35, except for a higher level of alanine aminotransferase and a lower level of Mg in the FFT group. Likewise, organ weights relative to body weight were largely similar on day 35, albeit with a lower stomach weight and more colon content in FFT relative to CON. Gut mucosal percentage and mucosal enzyme activity were similar between the two groups on days 27 and 35. Gut bacterial composition was slightly different on day 35 but not on day 27. In conclusion, early postnatal administration of FFT, showed positive clinical effects in post-weaning pigs, albeit with subtle effects on the gut mucosa and microbiome. Prophylactic treatment with FFT may offer a means to reduce morbidity, yet larger studies are required to document effect size.

Effects of early postnatal gastric and colonic microbiota transplantation on piglet gut health.

BACKGROUND: Diarrhea is a major cause of reduced growth and mortality in piglets during the suckling and weaning periods and poses a major threat to the global pig industry. Diarrhea and gut dysbiosis may in part be prevented via improved early postnatal microbial colonization of the gut. To secure better postnatal gut colonization, we hypothesized that transplantation of colonic or gastric content from healthy donors to newborn recipients would prevent diarrhea in the recipients in the post-weaning period. Our objective was to examine the impact of transplanting colonic or gastric content on health and growth parameters and paraclinical parameters in recipient single-housed piglets exposed to a weaning transition and challenged with enterotoxigenic Escherichia coli (ETEC). METHODS: Seventy-two 1-day-old piglets were randomized to four groups: colonic microbiota transplantation (CMT, n = 18), colonic content filtrate transplantation (CcFT, n = 18), gastric microbiota transplantation (GMT, n = 18), or saline (CON, n = 18). Inoculations were given on d 2 and 3 of life, and all piglets were milk-fed until weaning (d 20) and shortly after challenged with ETEC (d 24). We assessed growth, diarrhea prevalence, ETEC concentration, organ weight, blood parameters, small intestinal morphology and histology, gut mucosal function, and microbiota composition and diversity. RESULTS: Episodes of diarrhea were seen in all groups during both the milk- and the solid-feeding phase, possibly due to stress associated with single housing. However, CcFT showed lower diarrhea prevalence on d 27, 28, and 29 compared to CON (all P < 0.05). CcFT also showed a lower ETEC prevalence on d 27 (P < 0.05). CMT showed a higher alpha diversity and a difference in beta diversity compared to CON (P < 0.05). Growth and other paraclinical endpoints were similar across groups. CONCLUSION: In conclusion, only CcFT reduced ETEC-related post-weaning diarrhea. However, the protective effect was marginal, suggesting that higher doses, more effective modalities of administration, longer treatment periods, and better donor quality should be explored by future research to optimize the protective effects of transplantation.

Fecal virome transfer improves proliferation of commensal gut Akkermansia muciniphila and unexpectedly enhances the fertility rate in laboratory mice.

Probiotics are intended to improve gastrointestinal health when consumed. However, the probiotics marketed today only colonize the densely populated gut to a limited extent. Bacteriophages comprise the majority of viruses in the human gut virome and there are strong indications that they play important roles in shaping the gut microbiome. Here, we investigate the use of fecal virome transplantation (FVT, sterile filtrated feces) as a mean to alter the gut microbiome composition to lead the way for persistent colonization of two types of probiotics: Lacticaseibacillus rhamnosus GG (LGG) representing a well-established probiotic and Akkermansia muciniphila (AKM) representing a putative next-generation probiotic. Male and female C57BL/6NTac mice were cohoused in pairs from 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, LGG+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT donor material originated from mice with high relative abundance of A. muciniphila. All animals were terminated at age 9 weeks. The FVT treatment did not increase the relative abundance of the administered LGG or AKM in the recipient mice. Instead FVT significantly (p < 0.05) increased the abundance of naturally occurring A. muciniphila compared to the control. This highlights the potential of propagating the existing commensal "probiotics" that have already permanently colonized the gut. Being co-housed male and female, a fraction of the female mice became pregnant. Unexpectedly, the FVT treated mice were found to have a significantly (p < 0.05) higher fertility rate independent of probiotic administration. These preliminary observations urge for follow-up studies investigating interactions between the gut microbiome and fertility.

Donor-dependent fecal microbiota transplantation efficacy against necrotizing enterocolitis in preterm pigs.

The development of necrotizing enterocolitis (NEC), a life-threatening inflammatory bowel disease affecting preterm infants, is connected with gut microbiota dysbiosis. Using preterm piglets as a model for preterm infants we recently showed that fecal microbiota transplantation (FMT) from healthy suckling piglet donors to newborn preterm piglets decreased the NEC risk. However, in a follow-up study using donor stool from piglets recruited from another farm, this finding could not be replicated. This allowed us to study donor-recipient microbiota dynamics in a controlled model system with a clear difference in NEC phenotype. Preterm piglets (n = 38) were randomly allocated to receive control saline (CON), or rectal FMT using either the ineffective (FMT1) or the effective donor stool (FMT2). All animals were followed for four days before necropsy and gut pathological evaluation. Donor and recipient colonic gut microbiota (GM) were analyzed by 16 S rRNA gene amplicon sequencing and shotgun metagenomics. As expected, only FMT2 recipients were protected against NEC. Both FMT groups had shifted GM composition relative to CON, but FMT2 recipients had a higher lactobacilli relative abundance compared to FMT1. Limosilactobacillus reuteri and Lactobacillus crispatus strains of FMT recipients showed high phylogenetic similarity with their respective donors, indicating engraftment. Moreover, the FMT2 group had a higher lactobacilli replication rate and harbored specific glycosaminoglycan-degrading Bacteroides. In conclusion, subtle species-level donor differences translate to major changes in engraftment dynamics and the ability to prevent NEC. This could have implications for proper donor selection in future FMT trials for NEC prevention.

Glucose supply and glycolysis inhibition shape the clinical fate of Staphylococcus epidermidis-infected preterm newborns.

Preterm infants are susceptible to bloodstream infection by coagulase-negative staphylococci (CONS) that can lead to sepsis. Glucose-rich parenteral nutrition is commonly used to support the infants' growth and energy expenditure but may exceed endogenous regulation during infection, causing dysregulated immune response and clinical deterioration. Using a preterm piglet model of neonatal CONS sepsis induced by Staphylococcus epidermidis (S. epidermidis) infection, we demonstrate the delicate interplay between immunity and glucose metabolism to regulate the host infection response. Circulating glucose levels, glycolysis, and inflammatory response to infection are closely connected across the states of tolerance, resistance, and immunoparalysis. Furthermore, high parenteral glucose provision during infection induces hyperglycemia, elevated glycolysis, and inflammation, leading to metabolic acidosis and sepsis, whereas glucose-restricted individuals are clinically unaffected with increased gluconeogenesis to maintain moderate hypoglycemia. Finally, standard glucose supply maintaining normoglycemia or pharmacological glycolysis inhibition enhances bacterial clearance and dampens inflammation but fails to prevent sepsis. Our results uncover how blood glucose and glycolysis control circulating immune responses, in turn determining the clinical fate of preterm infants infected with CONS. Our findings suggest further refinements of the current practice of parenteral glucose supply for preterm infants during infection.

Differential Brain and Cerebrospinal Fluid Proteomic Responses to Acute Prenatal Endotoxin Exposure.

Chorioamnionitis (CA) is a risk factor for preterm birth and is associated with neurodevelopmental delay and cognitive disorders. Prenatal inflammation-induced brain injury may resolve during the immediate postnatal period when rapid brain remodeling occurs. Cerebrospinal fluid (CSF) collected at birth may be a critical source of predictive biomarkers. Using pigs as a model of preterm infants exposed to CA, we hypothesized that prenatal lipopolysaccharide (LPS) exposure induces proteome changes in the CSF and brain at birth and postnatally. Fetal piglets (103 days gestation of full-term at 117 days) were administered intra-amniotic (IA) lipopolysaccharide (LPS) 3 days before preterm delivery by caesarian section. CSF and brain tissue were collected on postnatal Days 1 and 5 (P1 and P5). CSF and hippocampal proteins were profiled by LC-MS-based quantitative proteomics. Neuroinflammatory responses in the cerebral cortex, periventricular white matter and hippocampus were evaluated by immunohistochemistry, and gene expression was evaluated by qPCR. Pigs exposed to LPS in utero showed changes in CSF protein levels at birth but not at P5. Complement protein C3, hemopexin, vasoactive intestinal peptide, carboxypeptidase N subunit 2, ITIH1, and plasminogen expression were upregulated in the CSF, while proteins associated with axon growth and synaptic functions (FGFR1, BASP1, HSPD1, UBER2N, and RCN2), adhesion (talin1), and neuronal survival (Atox1) were downregulated. Microglia, but not astrocytes, were activated by LPS at P5 in the hippocampus but not in other brain regions. At this time, marginal increases in complement protein C3, LBP, HIF1a, Basp1, Minpp1, and FGFR1 transcription indicated hippocampal proinflammatory responses. In conclusion, few days exposure to endotoxin prenatally induce proteome changes in the CSF and brain at birth, but most changes resolve a few days later. The developing hippocampus has high neuronal plasticity in response to perinatal inflammation. Changes in CSF protein expression at birth may predict later structural brain damage in preterm infants exposed to variable types and durations of CA-related inflammation in utero.

Fecal filtrate transplantation protects against necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disorder afflicting preterm infants, which is currently unpreventable. Fecal microbiota transplantation (FMT) is a promising preventive therapy, but the transfer of pathogenic microbes or toxic compounds raise concern. Removal of bacteria from donor feces by micropore filtering may reduce this risk of bacterial infection, while residual bacteriophages could maintain the NEC-preventive effects. We aimed to assess preclinical efficacy and safety of fecal filtrate transplantation (FFT). Using fecal material from healthy suckling piglets, we compared rectal FMT administration (FMT, n = 16) with cognate FFT by either rectal (FFTr, n = 14) or oro-gastric administration (FFTo, n = 13) and saline (CON, n = 16) in preterm, cesarean-delivered piglets as models for preterm infants. We assessed gut pathology and analyzed mucosal and luminal bacterial and viral composition using 16S rRNA gene amplicon and meta-virome sequencing. Finally, we used isolated ileal mucosa, coupled with RNA-Seq, to gauge the host response to the different treatments. Oro-gastric FFT completely prevented NEC, which was confirmed by microscopy, whereas FMT did not perform better than control. Oro-gastric FFT increased viral diversity and reduced Proteobacteria relative abundance in the ileal mucosa relative to control. An induction of mucosal immunity was observed in response to FMT but not FFT. As preterm infants are extremely vulnerable to infections, rational NEC-preventive strategies need incontestable safety profiles. We show in a clinically relevant animal model that FFT, as opposed to FMT, efficiently prevents NEC without any recognizable side effects.

Ultra-High Temperature Treatment and Storage of Infant Formula Induces Dietary Protein Modifications, Gut Dysfunction, and Inflammation in Preterm Pigs.

SCOPE: Ready-to-feed liquid infant formula is increasingly used for preterm infants when human milk is unavailable. These formulas are sterilized by ultra-high temperature treatment, but heating and storage may reduce bioactivity and increase formation of Maillard reaction products with potential negative consequences for immature newborns. METHODS AND RESULTS: Using preterm pigs as a model for sensitive newborn infants, the study tests the intestinal responses of feeding experimental liquid formula within 5 days. A pasteurized formula (PAST) with the same nutrient composition but less protein modifications serves as control to ultra-high temperature-treated formula without (UHT) and with prolonged storage (SUHT). Relative to PAST, UHT contains lower levels of lactoferrin and IgG. Additional storage (40 °C, 60 days, SUHT) reduces antimicrobial capacity and increases non-reducible protein aggregates and Maillard reaction products (up to 13-fold). Pigs fed SUHT have more diarrhea and show signs of intestinal inflammation (necrotizing enterocolitis) compared with pigs fed PAST and UHT. These clinical effects are accompanied by accumulation of Maillard reaction products, protein cross-links, and inflammatory responses in the gut. CONCLUSION: The results demonstrate that feeding UHT infant formulas, particularly after prolonged storage, adversely affects gut maturation and function in preterm pigs used as a model of preterm infants.

Co-bedding of Preterm Newborn Pigs Reduces Necrotizing Enterocolitis Incidence Independent of Vital Functions and Cortisol Levels.

Background: Preterm infants are born with immature organs, leading to morbidities such as necrotizing enterocolitis (NEC), a gut inflammatory disease associated with adverse feeding responses but also hemodynamic and respiratory instability. Skin-to-skin contact including "kangaroo care" may improve infant survival and health via improved vital functions (e.g., pulmonary, cardiovascular) and endocrine influences by adrenal glucocorticoids. Clinical effects of skin-to-skin contact for newborn siblings ("co-bedding") are not known. Using NEC-susceptible Preterm pigs as models, we hypothesized that co-bedding and exogenous glucocorticoids improve vital functions and NEC resistance. Methods: In experiment 1, cesarean-delivered, formula-fed Preterm pigs were reared in incubators with (co-bedding, COB, n = 30) or without (single-bedding, SIN, n = 29) a sibling until euthanasia and tissue collection on day four. In experiment 2, single-bedded Preterm pigs were treated postnatally with a tapering dose of hydrocortisone (HC, n = 19, 1-3 mg/kg/d) or saline (CON, n = 19). Results: Co-bedding reduced NEC incidence (38 vs. 65%, p < 0.05) and increased the density of colonic goblet cells (+20%, p < 0.05) but had no effect on pulmonary and cardiovascular functions (respiration, blood pressure, heart rate, blood gases) or cortisol levels. There were limited differences in intestinal villous architecture and digestive enzyme activities. In experiment 2, HC treatment increased NEC lesions in the small intestine without any effects on pulmonary or cardiovascular functions. Conclusion: Co-bedding may improve gut function and NEC resistance independently of cardiorespiratory function and cortisol levels, but pharmacological cortisol treatment predispose to NEC. Preterm pigs may be a useful tool to better understand the physiological effects of co-bedding, neonatal stressors and their possible interactions with morbidities in Preterm neonates.

Enteral broad-spectrum antibiotics antagonize the effect of fecal microbiota transplantation in preterm pigs.

Preterm infants are at risk of multiple morbidities including necrotizing enterocolitis (NEC). Suspected NEC patients receive intravenous antibiotics (AB) to prevent sepsis, although enteral AB is arguably more effective at reducing NEC but is rarely used due to the risk of AB resistance. Fecal microbiota transplantation (FMT) has shown protective effects against NEC in animal experiments, but the interaction between AB and FMT has not been investigated in neonates. We hypothesized that administration of enteral AB followed by rectal FMT would effectively prevent NEC with negligible changes in AB resistance and systemic immunity. Using preterm piglets, we examined host and gut microbiota responses to AB, FMT, or a sequential combination thereof, with emphasis on NEC development. In a saline-controlled experiment, preterm piglets (n = 67) received oro-gastric neomycin (50 mg/kg/d) and amoxicillin-clavulanate (50/12.5 mg/kg/d) (hereafter AB) for four days after cesarean delivery, and were subsequently given rectal FMT from healthy suckling piglet donors. Whereas AB protected the stomach and small intestine, and FMT primarily protected the colon, the sequential combination treatment surprisingly provided no NEC protection. Furthermore, minor changes in the gut microbiota composition were observed in response to either treatment, although AB treatment decreased species diversity and increased AB resistance among coliform bacteria and Enterococci, which were both partly reversed by FMT. Besides, enteral AB treatment suppressed cellular and functional systemic immune development, which was not prevented by subsequent FMT. We discovered an antagonistic relationship between enteral AB and FMT in terms of NEC development. The outcome may depend on choice of AB compounds, FMT composition, doses, treatment duration, and administration routes, but these results challenge the applicability of enteral AB and FMT in preterm infants.

Impaired Neonatal Immunity and Infection Resistance Following Fetal Growth Restriction in Preterm Pigs.

Background: Infants born preterm or small for gestational age (SGA, due to fetal growth restriction) both show an increased risk of neonatal infection. However, it remains unclear how the co-occurrence of preterm birth and SGA may affect neonatal immunity and infection risk. We hypothesized that fetal growth restricted (FGR) preterm newborns possess impaired immune competence and increased susceptibility to systemic infection and sepsis, relative to corresponding normal birth weight (NBW) newborns. Methods: Using preterm pigs as a model for preterm infants, gene expression in lipopolysaccharide (LPS) stimulated cord blood was compared between NBW and FGR (lowest 25% birth weight percentile) preterm pigs. Next, clinical responses to a systemic Staphylococcus epidermidis (SE) challenge were investigated in newborn FGR and NBW preterm pigs. Finally, occurrence of spontaneous infections were investigated in 9 d-old FGR and NBW preterm pigs, with or without neonatal antibiotics treatment. Results: At birth, preterm FGR piglets showed diminished ex vivo cord blood responses to LPS for genes related to both innate and adaptive immunity, and also more severe septic responses following SE infection (e.g., higher blood lactate, decreased blood pH, neutrophil and platelet counts, relative to NBW pigs). After 9 d, FGR pigs had higher incidence and severity of spontaneous infections (e.g., higher bacterial densities in the bone marrow), increased regulatory T cell numbers, reduced neutrophil phagocytosis capacity, and impaired ex vivo blood gene responses to LPS, especially when receiving neonatal antibiotics. Conclusion: FGR at preterm birth is associated with poor immune competence, impaired infection resistance, and greater sepsis susceptibility in the immediate postnatal period. Our results may explain the increased morbidity and mortality of SGA preterm infants and highlight the need for clinical vigilance for this highly sensitive subgroup of preterm neonates.

Diet Modulates the High Sensitivity to Systemic Infection in Newborn Preterm Pigs.

Background: Preterm infants are born with an immature immune system, limited passive immunity, and are at risk of developing bacteremia and sepsis in the postnatal period. We hypothesized that enteral feeding, with or without added immunoglobulins, improves the clinical response to systemic infection by coagulase negative staphylococci. Methods: Using preterm cesarean delivered pigs as models for preterm infants, we infused live Staphylococcus epidermidis (SE, 5 × 109 colony forming units per kg) systemically 0-3 days after birth across five different experiments. SE infection responses were assessed following different gestational age at birth (preterm vs. term), enteral milk diets (bovine colostrum, infant formula with or without added porcine plasma) and with/without systemic immunoglobulins. Pigs infected with SE were assessed 12-48 h for clinical variables, blood bacteriology, chemistry, hematology, and gut dysfunction (intestinal permeability, necrotizing enterocolitis lesions). Results: Adverse clinical responses and increased mortality were observed in preterm vs. term pigs, when infected with SE just after birth. Feeding bovine colostrum just after birth improved blood SE clearance and clinical status (improved physical activity and intestinal structure, fewer bone marrow bacteria), relative to pigs fed infant formula. A few days later, clinical responses to SE bacteremia (hematology, neutrophil phagocytic capacity, T cell subsets) were less severe, and less affected by different milk diets, with or without added immunoglobulins. Conclusion: Prematurity increases the sensitivity of newborn pigs to SE bacteremia, potentially causing sepsis. Sensitivity to systemic SE infection decreases rapidly in the days after preterm birth. Both age and diet (parenteral nutrition, colostrum, milk, formula) may influence gut inflammation, bacterial translocation and systemic immune development in the days after birth in preterm newborns.