Antimicrobial protein and Peptide concentrations and activity in human breast milk consumed by preterm infants at risk of late-onset neonatal sepsis

Abstract

Objective: We investigated the levels and antimicrobial activity of antimicrobial proteins and peptides (AMPs) in breast milk consumed by preterm infants, and whether deficiencies of these factors were associated with late-onset neonatal sepsis (LOS), a bacterial infection that frequently occurs in preterm infants in the neonatal period.

Study design: Breast milk from mothers of preterm infants (≤ 32 weeks gestation) was collected on days 7 (n = 88) and 21 (n = 77) postpartum. Concentrations of lactoferrin, LL-37, beta-defensins 1 and 2, and alpha-defensin 5 were measured by enzyme-linked immunosorbent assay. The antimicrobial activity of breast milk samples against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae was compared to the activity of infant formula, alone or supplemented with physiological levels of AMPs. Samples of breast milk fed to infants with and without subsequent LOS were compared for levels of AMPs and inhibition of bacterial growth.

Results: Levels of most AMPs and antibacterial activity in preterm breast milk were higher at day 7 than at day 21. Lactoferrin was the only AMP that limited pathogen growth >50% when added to formula at a concentration equivalent to that present in breast milk. Levels of AMPs were similar in the breast milk fed to infants with and without LOS, however, infants who developed LOS consumed significantly less breast milk and lower doses of milk AMPs than those who were free from LOS.

Conclusions: The concentrations of lactoferrin and defensins in preterm breast milk have antimicrobial activity against common neonatal pathogens.

Figure 1. Concentrations of soluble factors measured in breast milk.

Concentrations of LF (A); HBD1 (B); HBD2 (C); HD5 (D); LL-37 (E); and total protein (F), measured in day 7 (n = 88) and day 21 (n = 77) breast milk samples using ELISA or BCA assay for protein. All data are shown on a log scale with line at median. **p <0.01, ***p<0.001; comparing levels at 7 and 21 days postpartum using Wilcoxon signed-rank tests.

Figure 2. Bacterial growth-inhibition activities of breast milk samples.

Colony-forming units of (A) S. epidermidis; (B) S. aureus; (C) E. coli; or (D) S. agalactiae, after 4 h incubation in either LBWF (F; n = 16), day 7 (n = 40) or day 21 (n = 31) skimmed preterm breast milk samples from participants in the case-control study. The dashed line shows median starting inoculum. Data show individual and median values on a log scale. A value of 10³ CFU/mL was assigned to samples where the colony count was below the limit of detection of the assay. Symbols depict the groups where statistically significant comparisons were made (level of significance indicated by multiple symbols; e.g. *p <0.05, **p<0.01, ***p<0.001), comparing growth in LBWF to growth in preterm breast milk samples by ANOVA with Dunn’s multiple comparison test (*) or comparing growth in day 7 and day 21 paired breast milk samples by Wilcoxon signed-rank tests (†).

Figure 3. Inhibition of bacterial growth in LBWF spiked with antimicrobial proteins and peptides (AMPs).

Results from two experiments show median log-transformed colony-forming units (±interquartile range; n = 4) on a log scale of remaining CFU/mL from an inoculum of ~1 × 10⁶ of: (A) S. epidermidis; (B) S. aureus; (C) E. coli; or (D) S. agalactiae after 4 h incubation in LBWF (diagonal lined pattern) spiked with either no AMP (F; n = 8 shows both controls combined, statistical tests were performed using values from relevant day only), or with the low (L), median (M) or high (H) concentration of LF, HBD1, HBD2 or HD5 detected in breast milk samples. C = cocktail of LF, HBD1, HBD2 and HD5 at median concentrations (horizontal lined pattern). Shaded area indicates range of starting inoculum. Symbols depict the groups where statistically significant comparisons were made on log-transformed data (level of significance indicated by multiple symbols; *p <0.05, **p<0.01, ***p<0.001) in ANOVA with Dunnett’s multiple comparison tests of all high, median and low spiked treatments to LBWF control (*), or in a t-test of median LF to cocktail of LF plus other molecules (†).

Figure 4. Iron-dependence of inhibitory activity of lactoferrin-spiked LBWF or breast milk against LOS pathogens.

Bars show log-transformed medians and interquartile ranges from four replicates of bacterial colonies (Log CFU/mL) of (A) S. epidermidis; (B) S. aureus; (C) E. coli; or (D) S. agalactiae, after 4 h growth in LBWF control (shaded bar(“F”)), or LBWF or skimmed preterm breast milk (“M”) ± lactoferrin (“LF”; 3.8 mg/mL) and/or 1 mM ferric citrate (“Fe”). F control shows combined results of two separate experiments, however, treatment was statistically compared to experimental results on that day only. Symbols depict the groups where statistically significant comparisons were made on log-transformed data (level of significance indicated by multiple symbols; e.g. *p <0.05, **p<0.01, ***p<0.001), where (*) shows treatment compared to LBWF control after Dunnett’s multiple comparison test; (†) shows a significant result from an unpaired t-test comparing lactoferrin spiked LBWF ± ferric citrate; and (‡) shows a significant result from a paired t-test comparing four skimmed breast milks ± ferric citrate.

Figure 5. Consumption of breast milk (MOM+PDHM) by preterm infants in the case-control study.

Boxplots show median and interquartile ranges of (A) daily breast milk consumption and (B) cumulative milk dose (mL/kg) consumed by preterm infants in the nested case-control study from days 1–14 and 1–28, respectively, showing non-LOS (clear boxes, n = 20) and LOS infants (shaded boxes, n = 20), comparing cases and controls using Wilcoxon matched pairs analysis. Symbols indicate *p<0.05; **p<0.01.

Figure 6. Consumption of AMPs at day 7 and 21 postpartum in case-control infants.

Data show calculated consumed dose of (A) LF and (B) HBD1 in LOS infants (closed circles) or non-LOS infants (open circles) in the case-control study at day 7 (n = 19 each) and day 21 postpartum (n = 14 and n = 17, respectively), based on measured concentrations of AMPs in breast milk and reported milk consumption for each infant not consuming PDHM at the two time points on a log scale. Zero values calculated at day 7 (6 LOS, 7 non-LOS) and day 21 (4 LOS, zero non-LOS) were assigned a value of 0.01 for illustration purposes on the log scale. *p<0.05 comparing matched pairs.