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

Abstract

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.

Keywords: bacteremia; diet; feeding; immunity; infant; passive; preterm; sepsis.

Figure 1

Preterm birth increases susceptibility to Staphylococcus epidermidis bacteremia. Results of Experiment 1 comparing term (TERM) and preterm (PRE) pigs infused with Staphylococcus epidermidis (SE) or saline (CON) immediately after birth, without any enteral feeding. (A) Survival rates 24 h post inoculation. (B) Motor activity, shown as the fraction of time when pigs were physically moving at 2–18 h post inoculation. (C) Body temperature 2–24 h post inoculation. (D) Blood pH 6–24 h post inoculation. (E) Blood lactate levels 6–24 h post inoculation. (A) Presented as Kaplan Meyer curves. (B–E) Presented as means with corresponding standard errors. (#): Effect of SE (p ≤ 0.1 ≥ 0.05), #: Effect of SE (p < 0. 05), ###: Effect of SE (p < 0.001), ¤: Effect of gestational age (p < 0.05).

Figure 2

Feeding of bovine colostrum improves clearance of Staphylococcus epidermidis in newborn preterm pigs. Results of Experiment 2 comparing preterm pigs infused with Staphylococcus epidermidis (SE) immediately after birth and fed either bovine colostrum or infant formula. (A) Abundance of SE in blood 3–12 h post inoculation, shown as colony-forming units per milliliter of whole blood. (B) Blood lactate levels 3-12 h post inoculation. (C) Blood bicarbonate levels 3–12 h post inoculation. (D) Blood pH 3–12 h post inoculation. (E) Total leucocyte, neutrophil and lymphocyte counts, taken 12 h post inoculation. (A–E) Presented as means with corresponding standard errors. (*): Effect of diet (p ≤ 0.1 ≥ 0.05), *Effect of diet (p < 0.05).

Figure 3

Two days of bovine colostrum feeding has no impact on response to Staphylococcus epidermidis bacteremia. Results of Experiment 3 comparing preterm pigs infused with Staphylococcus epidermidis (SE) or saline (CON) at day 3, after being infused with maternal plasma and fed bovine colostrum (BC) or infant formula (IF) from after birth. (A) Body temperature 0–48 h post inoculation. (B) Motor activity, shown as the fraction of time with physical activity at 0–48 h post inoculation. (C) Neutrophil phagocytic rate 12–48 h post inoculation, defined as fraction of neutrophils with internalized bacteria. (D) Neutrophil phagocytic capacity 12–48 h post inoculation, defined as median fluorescent index of neutrophils with internalized bacteria. (E) T cells 12–48 h post inoculation, defined as fraction of CD3 positive lymphocytes. (F) CD4 cells 12–48 h post inoculation, defined as the CD4 positive, CD8 and FOXP3 negative fraction of T cells. (G) CD8 cells 12–48 h post inoculation, defined as the CD8 positive, CD4 and FOXP3 negative fraction of T cells. (H) Regulatory T cells 12–48 h post inoculation, defined as the FOXP3 and CD4 positive, CD8 negative fraction of T cells. CD4 positive as fraction (I) Plasma levels of bovine immunoglobulin G (IgG) at euthanasia. (A–I) Presented as means with corresponding standard errors. #: Effect of SE (p < 0.05), ##: Effect of SE (p < 0.01), ###: Effect of SE (p < 0.001), (*): Effect of diet (p ≤ 0.1 ≥ 0.05), *: Effect of diet (p < 0.05), **: Effect of diet (p < 0.01), ***: Effect of diet (p < 0.001).

Figure 4

Two days of bovine colostrum feeding has no impact on response to Staphylococcus epidermidis bacteremia, even when maternal antibodies are withheld. Results of Experiment 4 where preterm pigs were infused with Staphylococcus epidermidis (SE) on day 3, without any previous immunization (plasma infusion) and fed bovine colostrum (SE-BC) or infant formula (SE-IF). (A) Body temperature 0–24 h post inoculation. (B) Motor activity, shown as the fraction of time with physical activity at 0–24 h post inoculation. (C) Blood pH 6–24 h post inoculation. (D) Blood oxygen pressure 6–24 h post inoculation. (C) Blood bicarbonate levels 6–24 h post inoculation. (F) Plasma levels of tumor necrosis factor alpha (TNF-α) 6–24 h post inoculation. (G) Plasma levels of soluble terminal complement complexes (sC5b-9) 6–12 h post inoculation. (H) Abundance of SE in blood at euthanasia, shown as colony-forming units per milliliter of whole blood. (I) Plasma levels of bovine immunoglobulin G (IgG) at euthanasia. (A–F) Presented as means with corresponding standard errors. *: Effect of diet (p < 0.05), **: Effect of diet (p < 0.01), ***: Effect of diet (p < 0.001).

Figure 5

Feeding of infant formula, enriched with porcine plasma, has no effect on responses to Staphylococcus epidermidis bacteremia. Results of Experiment 5 where preterm pigs were infused with Staphylococcus epidermidis (SE) on day 4, after infusion of maternal plasma at birth, and then fed infant formula supplemented with porcine plasma powder (SE-PP) or whey protein (SE-IF). (A) Body temperature 0–24 h post inoculation. (B) Motor activity, shown as the fraction of time with physical movements at 0–24 h post inoculation. (C) Total blood leucocyte counts 0–24 h post inoculation. (D) Lymphocyte counts in blood, 0–24 h post inoculation (E) Abundance of SE in blood at euthanasia, shown colony-forming units per milliliter of whole blood. (F) Plasma levels of porcine immunoglobulin G (IgG) at euthanasia. (A–F) Presented as means with corresponding standard errors. ϕ: Effect of PP (p < 0.05).