Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus

Abstract

Streptococcus agalactiae (Group B Streptococcus, GBS) is a Gram-positive bacterial pathogen that causes invasive infections in both children and adults. During pregnancy, GBS is a significant cause of infection of the fetal membranes (chorioamnionitis), which can lead to intra-amniotic infection, preterm birth, stillbirth, and neonatal sepsis. Recently, breastfeeding has been thought to represent a potential mode of GBS transmission from mother to newborn, which might increase the risk for late-onset sepsis. Little is known, however, about the molecular components of breast milk that may support or prevent GBS colonization. In this study, we examine how human milk oligosaccharides (HMOs) affect the pathogenesis of GBS. HMOs from discrete donor samples were isolated and profiled by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Growth and biofilm assays show that HMOs from mothers of specific milk groups can modulate the growth and biofilm formation of GBS. High-resolution field-emission gun scanning electron microscopy (SEM) and confocal laser scanning microscopy confirmed the quantitative biofilm assays and demonstrated cell arrangement perturbations in bacterial cultures treated with specific oligosaccharides. These findings demonstrate that HMOs affect the growth and cell biology of GBS. Finally, this study provides the first example of HMOs functioning as antibiofilm agents against GBS.

Keywords: GBS; Group B Streptococcus; HMO; antibiofilm; antimicrobial; bacteriostatic; human milk oligosaccharides.

Figure 1

Effects of human milk oligosaccharides on Group B Streptococccus. A. Previous research (Bode, 2015 and 2017; Le Doare, 2016): HMOs act as antibacterial agents against GBS B. This work: HMOs from donors assigned specific Lewis blood groups act as bacteriostatic and anti-biofilm agents

Figure 2

MALDI-FT-ICR-MS/MS spectra of (A) m/z 657.2 and (B) m/z 1022.2 from 700-900 of HMOs from 5 separate donors obtained in the positive ion mode. Diagnostic peaks highlighted in red.

Figure 3

Effect of HMOs isolated from individual milk samples on growth rate/proliferation of GBS 10/84 in Todd Hewitt Broth (A) OD₆₀₀ readings were taken at 0, 2-12, 22, and 24 h. Mean OD₆₀₀ for each HMO sample and time point is indicated by the respective symbols. (B) Enumeration of CFU was performed at 0, 2-12, 22, and 24 h, corresponding to the OD values graphed in panel A. The mean CFU/mL was calculated for each time point and is indicated by the respective symbols. Data displayed represents the mean OD +/− SEM of 3 biological replicates, * P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001 by 2-way ANOVA with post-hoc Dunnett’s mutiple comparison test, with all donor samples compared to the GBS growth in media alone.

Figure 4

Effect of HMOs isolated from individual milk samples on growth rate/proliferation of GBS 10/84 in Todd Hewitt Broth supplemented with 1% glucose (A) OD₆₀₀ readings were taken at 0, 2-12, 22, 24, and 26 h. Mean OD₆₀₀ for each HMO sample and time point is indicated by the respective symbols. (B) Enumeration of CFU was performed at 0, 2-12, 22, 24, and 26 h, corresponding to the OD values graphed in panel A. The mean CFU/mL was calculated for each time point and is indicated by the respective symbols. Data displayed represents the mean OD +/− SEM of 3 biological replicates, * P<0.05, ** P<0.01, *** P<0.001, ****P<0.0001 by 2-way ANOVA with post-hoc Dunnett’s mutiple comparison test, with all donor samples compared to the GBS growth in media alone.

Figure 5

HMOs at biologically relevant breast milk concentrations (4.88 mg/mL) induce changes in biofilm formation of GBS cultures. The total biofilm to biomass ratio after 24 hours of growth was compared for (A) THB medium alone. Data represented as the mean biofilm/biomass ratio +/− SEM of 5 separate experiments, each with 3 technical replicates. *** represents p = 0.0008 by one-way ANOVA, F = 23.35 with post-hoc Dunnet’s multiple comparison test comparing each HMO group against the control sample without HMOs. (B) THB medium supplemented with 1% glucose. Data are expressed as the mean biofilm/biomass ratio +/− SEM of 5 separate experiments, each with 3 technical replicates. ** represents p = 0.0018 by one-way ANOVA, F = 3.449 with post-hoc Dunnet’s multiple comparison, compared to media alone. (C) Biofilm measurements of GBS grown in THB medium and (D) THB medium supplemented with 1% glucose. Data are expressed as the mean biofilm measurments (OD₅₆₀) +/− SEM of 5 separate experiments, each with 3 technical replicates. * represents p = < 0.05 by one-way ANOVA, F = 5.935 with post-hoc Dunnet’s multiple comparison, compared to media alone. (E) Average measurement of biofilm quantities represented by optical density (OD₅₆₀) of 5 separate experiments, each with 3 technical replicates. IC₅₀ values are listed for donors that inhibited growth. * represents p = < 0.05 by one-way ANOVA, F = 5.935 with post-hoc Dunnet’s multiple comparison, compared to media alone.

Figure 6

Scanning electron micrographs of biofilm formation after 24 h. GBS 10/84 cells were grown in THB + 1% glucose supplemented with individual donor samples for 24 hours at 37°C. Images are shown at ×250 magnification.

Figure 7

Scanning electron micrographs of biofilm formation after 24 h. GBS 10/84 cells were grown in THB + 1% glucose supplemented with HMOs from individual donor samples for 24 hours at 37°C. Images are shown at ×1,000 magnification.

Figure 8

CLSM micrographs comparing biofilm formation of GBS 10/84 grown in THB supplemented with 1% glucose or THB supplemented with 1% glucose and HMOs isolated from milk donors. Bacteria were grown under static conditions at 37°C for 24 hours on glass coverslips. Biofilms were stained immediately prior to analysis with SYTO-9 (green, live bacterial cells), propidium iodide (red, dead bacterial cells), and Calcofluor White (blue, carbohydrates) at 600× magnification. Images shown represent a z-stack series of images of the three stains where the larger panel is a “bird´s eye” view of the biofilms and the right and upper panels are side views of the x- and y-axis sections respectively.

Figure 9

CLSM micrographs comparing apical and base sections of GBS 10/84 biofilms grown in THB supplemented with 1% glucose or THB supplemented with 1% glucose and HMOs isolated from milk donors. Bacteria were grown under static conditions at 37°C for 24 hours on glass coverslips. Images shown represent the apical surface (left image) and base of biofilm (right image) from a z-stack series. Biofilms were stained with SYTO-9 (green, live bacterial cells), propidium iodide (red, dead bacterial cells), and Calcofluor White (blue, carbohydrates) and imaged at 600× magnification.

Figure 10

HMOs at physiologial breast milk concentrations (4.88 mg/mL) coupled with AMPs decrease growth and biofilm formation of GBS cultures. (A) Growth of GBS (OD₆₀₀) and (B) biofilm measurments of GBS after 24 hours in THB medium with increasing concentrations of Polymixin B. (C) Growth of GBS (OD₆₀₀) and (D) biofilm measurments of GBS after 24 hours in THB medium supplemented with 1% glucose and increasing concentrations of Polymixin B. Data represented as the mean biofilm/biomass ratio +/− SEM of 3 separate experiments, each with 3 technical replicates.