Anti-Pneumococcal Properties of the Native Human Milk Oligosaccharide Fraction: A Concentration-Dependent Study

<i>Streptococcus pneumoniae</i> is a major opportunistic pathogen and a leading cause of severe infections in infants under two years of age. Human milk oligosaccharides (HMOs), key bioactive components of breast milk, possess immunomodulatory and antimicrobial properties. In this study, the antipneumococcal effects of HMOs are investigated across multiple <i>S. pneumoniae</i> serotypes, focusing on concentration-dependent activity and underlying mechanisms. Growth inhibition and bacterial viability were evaluated using growth curve analysis and colony-forming unit (CFU) assays. HMOs inhibited pneumococcal growth in a concentration-dependent manner, with suppression observed at 1.5-2.5 mg/mL and complete killing at 5 mg/mL for all serotypes. Nonencapsulated strains were more sensitive, with inhibition at 1 mg/mL. In the CFU assays, killing occurred at 1.25-5 mg/mL depending on the strain. At physiologically relevant colostrum concentrations (20-25 mg/mL), HMOs achieved complete bactericidal effects across all the tested strains. In contrast, lactose at equivalent doses showed no measurable antimicrobial activity, confirming the specificity of the observed effects. Overall, HMOs exhibit serotype-independent antipneumococcal activity, possibly through interference with bacterial adhesion or metabolic disruption. These findings suggest a potential role for HMOs as adjunctive agents in the prevention of pneumococcal infections in vulnerable populations, such as infants, and warrant further in vivo studies to validate these effects and explore clinical applications.