HAMLET, a human milk protein-lipid complex, modulates amoxicillin-induced changes in an ex vivo biofilm model of the oral microbiome

Challenges from infections caused by biofilms and antimicrobial resistance highlight the need for novel antimicrobials that work synergistically with antibiotics and minimize resistance risk. In this study we investigated the potential synergistic effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells), a human milk protein-lipid complex and amoxicillin on microbial ecology using an ex-vivo oral biofilm model. HAMLET was chosen due to its multi-targeted antimicrobial mechanism, together with its synergistic effect with antibiotics on single species pathogens, and low risk of resistance development. The combination of HAMLET and amoxicillin significantly reduced biofilm viability, while each of them alone had little or no impact. Using a whole metagenomics approach, we found that the combination group promoted a most remarkable shift in overall microbial composition compared to the untreated samples. Up to 90% of the bacterial species in the combined treatment were Lactobacillus crispatus, a species with probiotic effects, whereas it was detected in minor fraction in untreated samples. Resistome analysis indicated no major shifts on alpha-diversity, while beta-diversity revealed distinct clustering patterns for each treatment group, signifying that each treatment group harbors a unique resistome. TEM beta-lactamase genes were detected in low proportions in all treated samples but absent in untreated samples. Our study highlights the potential of HAMLET to synergize with amoxicillin in an ex-vivo model of the oral microbiome and modulate the proportion of probiotic bacteria. The findings extend the knowledge on the synergistic effects of HAMLET and antibiotics from single-species studies to polymicrobial biofilms of human origin.