Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus

Abstract

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.

Keywords: Streptococcus; antimicrobial; biofilms; innate immunity; lactoferrin.

Figure 1.. Purification of the innate immune antimicrobial glycoprotein, lactoferrin.

A) Conceptual diagram of the protocol for purification of lactoferrin from human breast milk. Milk is centrifuged, lipid layer is removed, ethanol is added to precipitate milk proteins under refrigeration, and lactoferrin is purified by chromatography techniques. B) Chromatograph indicating a single peak, demonstrating purity of lactoferrin sample. C) Mass spectrometry results demonstrating the identity of lactoferrin within the sample. D) Circular dichroism graph results demonstrating the proper folding of lactoferrin, and that the bulk of the sample is present in the apo-form.

Figure 2.. Analysis of bacterial growth in increasing concentrations of lactoferrin.

GBS was grown in increasing concentrations (0, 250, 500, 750, 1000 μg/mL) of A) apo-lactoferrin (apo-Lf) or B) holo-lactoferrin (holo-Lf) over a 24-hour period. Bacterial growth was determined by measuring cellular density (optical density at 600 nm or OD₆₀₀). Points equal a mean +/− SEM, n=3-5. *P<0.05, Student’s t test comparison to bacteria grown in medium alone at the same time point. Apo-lactoferrin significantly inhibits GBS growth at concentrations of 500, 750, and 1000 μg/mL at 3, 4, 5, 6, 7, 8, 9, and 10 hours post-inoculation, a result that was ablated by saturation of the glycoprotein with iron.

Figure 3.. Analysis of in vitro bacterial biofilm formation on an abiotic surface in the presence or absence of lactoferrin.

GBS was grown in medium alone or medium supplemented with 250 μg/mL of either apo-lactoferrin (apo-Lf) or holo-lactoferrin (holo-Lf). Bacterial biofilm was analyzed by A) high-resolution scanning electron microscopy (SEM) at low magnification (2,500x, magnification bar indicates 50 μm) and high magnification (10,000x, magnification bar indicates 10 μm) and by B) quantitative analysis of crystal violet staining at an optical density of 560 nm (OD₅₆₀) normalized to bacterial cell density (OD₆₀₀). Bars indicate mean values of at least three biological replicates +/−SEM. *P<0.05, Student’s t test comparison to bacteria grown in medium alone. Apo-lactoferrin significantly inhibits GBS biofilm formation, while holo-lactoferrin has an intermediate phenotype that is statistically indistinguishable from the negative control.

Figure 4.. Analysis of ex vivo bacterial biofilm formation on an abiotic surface in the presence or absence of lactoferrin.

GBS was grown in co-culture with gestational membrane tissue in medium alone or medium supplemented with 250 μg/mL of either apo-lactoferrin (apo-Lf) or holo-lactoferrin (holo-Lf). Bacterial biofilm was analyzed by either immunohistochemical (IHC) staining with an antibody specifically to GBS (brown staining) and high-resolution scanning electron microscopy (SEM) at high magnification (10,000x, magnification bar indicates 10 μm). Apo-lactoferrin significantly inhibits GBS adherence to gestational membrane tissue, while holo-lactoferrin has an intermediate phenotype that is comparable to the control sample cultured in medium alone.

Figure 5.. Model of lactoferrin iron binding-dependent inhibition of biofilm and growth.

Free iron ions promote GBS biofilm formation. Apo-lactoferrin binds iron and inhibits biofilm formation and GBS growth via iron sequestration in a process termed, “nutritional immunity”. Apo-lactoferrin binds iron and inhibits GBS adherence to gestational membranes. Both holo- and apo-lactoferrin have the capacity to repress GBS viability.