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

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

Comparison of the total cell concentrations obtained from milk samples with different methods.

<p>Figure shows scatterplot of total cell counts obtained through microscopy on x-axis, compared with total CD45+ leukocyte concentrations obtained with flow cytometry on the y-axis. All data are shown on a log scale.</p

Changes to the composition of breast milk through the first month of lactation.

<p>Line shows the median value for that milk sampling time point. Data show A) concentrations of total CD45+ cells in colostrum (C), transitional milk (TM) or mature milk (MM); B-L) frequencies of leukocyte subsets in colostrum (C), transitional milk (TM) and mature milk (MM) from all donors. *p<0.05 in post-test comparing different stages of lactation; for each comparison, number of symbols indicates p-value (*p<0.05, **p<0.01, ***p<0.001).</p

Overlay of gating strategy applied to peripheral adult blood (red) and to breast milk (blue), based on the method of Faucher <i>et al</i>.

<p>The sequences of gates leading to each panel are shown above each panel. Panel 1: CD45 positive cells were gated as shown in A. Panel 2: CD45+ cells identified in Panel 1 were separated based on CD16 staining and side scatter properties, including a C16+/SSClow gate (B), and two overlapping gates of CD16- cells (C and D), and the CD16+ neutrophil population were identified. Panel 3: CD45+/CD16+/SSClow cells identified in Panel 2 gate B were separated into cytotoxic T and NK lymphocytes and CD16+ monocytes based on CD2/CD294 and CD36 staining properties. Panel 4: CD45+/CD16-/SSClow–intermediate cells (gate C) were separated based on CD2/CD294 positive (gate E) or negative (gate F) populations. Panel 5: From Panel 4, CD2 and/or CD294 positive cells (gate E) were gated into non-cytotoxic T cells or basophils using side scatter and CD45 staining properties. Panel 6: From Panel 4, CD2/CD294- cells (gate F) were gated into CD19+/CD36- cells (gate H) or CD19- cells (gate G). Panel 7a: Cells gated in G in Panel 6 with CD45high and CD36+ were identified as CD16 negative monocytes. Panel 7b: From Panel 6 gate G, CD45low cells with low side scatter were identified as myeloid precursor cells. Panel 8: From Panel 6, CD19 positive cells (gate H) were discriminated into B cells or B cell precursors based on CD45 staining though both populations displayed low side scatter properties. Panel 9: From Panel 2, CD16- cells with intermediate to high side scatter (gate D) and CD45low staining were identified as immature granulocytes, and those with intermediate to high CD45 staining properties and high side scatter were separated into gate I. Panel 10: From Panel 9, cells in gate I were identified by positive CD2/CD294 staining as eosinophils.</p

Leukocyte concentrations in breast milk samples.

<p>Boxplots showing the total concentration of leukocytes (cells/mL) on a log scale in colostrum (C, n = 41), transitional milk (TM, n = 47), and mature milk (MM, n = 46), in extremely preterm (white), very preterm (light grey), moderately preterm (dark grey) and term (striped) donors.</p

Comparison of the total cell concentrations obtained from milk samples with different methods.

<p>Figure shows scatterplot of total cell counts obtained through microscopy on x-axis, compared with total CD45+ leukocyte concentrations obtained with flow cytometry on the y-axis. All data are shown on a log scale.</p

Comparison of clinical data for cases and controls used in the nested case-control study of breast milk antimicrobial molecules.

<p>Infant n = 20 in each group, however, at day 21, only 14 LOS infants and 17 non-LOS infants mothers’ provided milk. p<0.05 considered significant; shown in bold font.</p><p>^aMean±SD;</p><p>^bMedian (IQR);</p><p>^cProportion (%);</p><p>MOM = mothers’ own milk; PDHM = pasteurised donor human milk.</p><p>*Data were only collected on milk feeds to day 28, therefore infants who had not reached full doses by day 28 were designated as receiving milk at day 28 arbitrarily (n = 12 LOS, 5 non-LOS).</p><p>Comparison of clinical data for cases and controls used in the nested case-control study of breast milk antimicrobial molecules.</p

Bacterial growth-inhibition activities of breast milk samples.

<p>Colony-forming units of (A) <i>S. epidermidis</i>; (B) <i>S. aureus</i>; (C) <i>E. coli</i>; or (D) <i>S. agalactiae</i>, 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 (†).</p

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

<p>Bars show log-transformed medians and interquartile ranges from four replicates of bacterial colonies (Log CFU/mL) of (A) <i>S. epidermidis</i>; (B) <i>S. aureus</i>; (C) <i>E. coli</i>; or (D) <i>S. agalactiae</i>, 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.</p

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

<p>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.</p