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Antimicrobial Properties of Human Milk Oligosaccharides

 

By: Carol A. Rouzer, VICB Communications
Published: June 26, 2017

 

 

Oligosaccharides in human milk suppress growth and alter biofilm formation by Group B Streptococcus.

 

A rapidly growing body of evidence supports the benefits of human breast milk to infant health and development (Figure 1). Breast milk not only contains the ideal nutrient mix for human growth, it also provides multiple components that protect the infant from infection and promote appropriate immune system responses. Among these is a mixture of over 400 species of mostly symbiotic and commensal bacteria that ultimately establish the infant's intestinal microbiome. Yet, nursing is not completely risk free. Milk may contain potentially pathogenic organisms, such as Streptococcus agalactiae, also known as Group B Streptococcus (GBS), a Gram positive bacterium that can cause meningitis and sepsis in young infants. Such concerns led Vanderbilt Institute of Chemical Biology members Steven Townsend and Jennifer Gaddy along with their collaborator David Aronoff to explore the effects of human milk oligosaccharides (HMOs) on GBS growth and virulence. Their results show that HMOs may play a role in protecting infants from infection from GBS [D. L. Ackerman, et al., (2017) ACS Inf. Dis., published online June 1, 2017, DOI:10.1021/acsinfdis.7b00064 . Copyright 2017].

 

FIGURE 1. Image reproduced under the GNU Free Documentation License from Wikimedia Commons.

 

HMOs are the third most abundant macromolecular component of milk. Each individual HMO comprises only five monosaccharide residues, but over 200 unique structures have been characterized. The exact mixture of HMOs in a mother's milk depends largely on two genes that also determine her Lewis blood group and secretor status. The first gene, Le, encodes an enzyme that adds a fucose residue to the penultimate position of a precursor oligosaccharide, yielding the Le(a) antigen. The second gene, Se, encodes an enzyme that adds a fucose residue to the terminal position of Le(a) converting it to Le(b). For both Le and Se, there exists a dominant allele (designated Le and Se, respectively) that encodes the functional protein and a recessive allele (designated le and se) that encodes an inactive enzyme. Since Le(a) is the precursor for Le(b), people with the le/le genotype fail to express Le(a) or Le(b) regardless of their Se status. Those with the se/se genotype and at least one Le allele express Le(a). Those with at least one Le and one Se allele express Le(b), since nearly all Le(a) is converted to Le(b) by the Se gene product. People who carry at least one Se allele also secrete the A/B/H blood group antigens in body fluids and are termed secretors. Although traditionally recognized for their role in producing red blood cell-associated oligosaccharides, the Le- and Se-encoded enzymes also have a major impact on HMO biosynthesis as indicated by the fact that a woman's Lewis and secretor status can be elucidated by mass spectrometric analysis of the HMOs in her milk. The investigators began their study by carrying out this precise analysis using the HMO fractions isolated from milk provided by five donors. The results identified donor 1 as Le(a+b-), donors 2 and 4 as Le(a-b+), and donors 3 and 5 as Le(a-b-). Of the five women, all were secretors with the exception of donor 1.

 

Having characterized the HMO composition of the milk from each donor, the investigators next explored the effect of the individual HMO mixtures on the growth of GBS (Figure 2). They discovered that, when added to growth medium in the absence of any other supplements, HMOs from donor 1 exerted a substantial suppressive effect on bacterial growth. HMOs from donor 3 were also inhibitory, but with much lower potency than those from donor 1 (Figure 2A). When the medium was supplemented with 1% glucose, the inhibitory effect of HMOs from donor 3 disappeared, whereas those from donor 1 retained their suppressive effects but only for a limited time period (Figure 2B). Thus, it appears that donor 1 HMOs were bacteriostatic (suppress bacterial growth) rather than bactericidal (kill the bacteria).

 

 

 

FIGURE 2. Effect of HMOs from five different donors on LBS growth. Bacteria were grown in medium alone (A) or medium plus 1% glucose (B) in the presence of HBOs from the indicated donors. Bacterial growth was measured on the basis of increasing absorbance of light at 600 nm. Image reproduced with permission from D. L. Ackerman, et al., (2017) ACS Inf. Dis., published online June 1, 2017, DOI:10.1021/acsinfdis.7b00064. Copyright 2017 American Chemical Society.

 

 

An important property of many kinds of bacteria, including GBS, is their ability to form biofilms – thin layers of bacterial cells that coat a surface. Bacteria in biofilms become specialized for particular functions, creating a survival advantage for the biofilm as a whole. In fact, biofilm formation helps pathogenic bacteria evade host defenses and resist antibiotics, so biofilm formation contributes to virulence. To see if HMOs have the ability to modulate biofilm formation, the investigators grew GBS on a surface in the presence or absence of each of their five donor samples. In the absence of supplementary glucose, none of the HMOs affected the total mass of biofilm formed. HMOs from donor 1 increased the ratio of biofilm to total bacterial cells, but this was because of its suppressive effect on overall growth. When glucose was added to promote biofilm formation, the HMOs from donors 1 and 3 caused a small but significant decrease in total biofilm mass, but that translated into a decrease in the ratio of biofilm mass to total bacterial mass only for the HMOs from donor 3. More striking effects were evident in the case of biofilm structure and morphology. In particular, HMOs from donor 1 produced a shortening of the chains formed by the GBS, leading to smaller "mushroom" structures in the biofilm and a less diffuse overall pattern. HMOs from donors 3 and 5 appeared to reduce nutrient channel formation in the biofilm, and donor 3 HMOs also reduced the thickness of the film (Figures 3 through 5).

 

FIGURE 3. Scanning electron micrographs (250X) of biofilms formed after 24 h in the presence of medium supplemented with 1% glucose and the indicated donor HMOs. Image reproduced with permission from D. L. Ackerman, et al., (2017) ACS Inf. Dis., published online June 1, 2017, DOI:10.1021/acsinfdis.7b00064. Copyright 2017 American Chemical Society.

 

FIGURE 4. Scanning electron micrographs (1000X) of biofilms formed after 24 h in the presence of medium supplemented with 1% glucose and the indicated donor HMOs. Image reproduced with permission from D. L. Ackerman, et al., (2017) ACS Inf. Dis., published online June 1, 2017, DOI:10.1021/acsinfdis.7b00064.

 

FIGURE 5. Confocal laser scanning micrographs (600X) of biofilms formed after 24 h in the presence of medium supplemented with 1% glucose and the indicated donor HMOs. The biofilms were stained for carboyhydrate (blue), dead cells (red), and live cells (green). The images are a z-stack series of the three stains. The major panel is a view from above the biofim, and the strips at the right and top are side views. Image reproduced with permission from D. L. Ackerman, et al., (2017) ACS Inf. Dis., published online June 1, 2017, DOI:10.1021/acsinfdis.7b00064.

 

 

These results suggest that HMOs can, by themselves, modulate GBS growth and biofilm formation. The investigators next tested the hypothesis that HMOs might interact with naturally occurring antimicrobial peptides to affect bacterial growth. They found that, in the presence of the antimicrobial peptide polymixin B, the HMOs from donor 1 caused total inhibition of bacterial growth as long as no supplementary glucose was added to the medium. The addition of 1% glucose, however, eliminated this effect. Polymixin B alone caused a dose-dependent reduction in biofilm formation in both the presence and absence of supplementary glucose, but none of the HMOs tested modified Polymixin B's action in this assay.

 

Together, the findings demonstrate that HMOs have the capacity to modulate bacterial growth and virulence; however, this capacity is dependent upon the particular HMO structural composition. It is interesting that the most effective HMO sample was from the only donor who was Le(a+b-) and a nonsecretor, though the reason for this is currently unknown. The researchers hypothesize that alteration of bacterial carbohydrate metabolism in the presence of HMOs could lead to the production of toxic metabolites. Alternatively, the availability of large quantities of exogenous sugars might provide a nutrient excess that suppresses the need for biofilm formation. A possible third effect of HMOs might be interference with quorum sensing that bacteria use to communicate with one another during biofilm formation. Further research is clearly required to better understand the foundation for these very interesting observations.

 

 

ViewACS Infectious Diseases article: Human Milk Oligosaccharides Exhibit Antimicrobial and Antibiofilm Properties against Group B Streptococcus

 

 

 

 

 

 

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