A Simple Polymicrobial Biofilm Keratinocyte Colonization Model for Exploring Interactions Between Commensals, Pathogens and Antimicrobials

Abstract

Skin offers protection against external insults, with the skin microbiota playing a crucial defensive role against pathogens that gain access when the skin barrier is breached. Linkages between skin microbes, biofilms and disease have not been well established although single-species biofilm formation by skin microbiota <i>in vitro</i> has been extensively studied. Consequently, the purpose of this work was to optimize and validate a simple polymicrobial biofilm keratinocyte model for investigating commensal, pathogen and keratinocyte interactions and for evaluating therapeutic agents or health promoting interventions. The model incorporates the commensals (<i>Staphylococcus epidermidis</i> and <i>Micrococcus luteus</i>) and pathogens (<i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>) which form robust polymicrobial biofilms on immortalized keratinocytes (HaCat cells). We observed that the commensals reduce the damage caused to the keratinocyte monolayer by either pathogen. When the commensals were combined with <i>P. aeruginosa</i> and <i>S. aureus</i>, much thinner biofilms were observed than those formed by the pathogens alone. When <i>P. aeruginosa</i> was inoculated with <i>S. epidermidis</i> in the presence or absence of <i>M. luteus</i>, the commensals formed a layer between the keratinocytes and pathogen. Although <i>S. aureus</i> completely inhibited the growth of <i>M. luteus</i> in dual-species biofilms, inclusion of <i>S. epidermidis</i> in triple or quadruple species biofilms, enabled <i>M. luteus</i> to retain viability. Using this polymicrobial biofilm keratinocyte model, we demonstrate that a quorum sensing (QS) deficient <i>S. aureus agr</i> mutant, in contrast to the parent, failed to damage the keratinocyte monolayer unless supplied with the exogenous cognate autoinducing peptide. In addition, we show that treatment of the polymicrobial keratinocyte model with nanoparticles containing an inhibitor of the PQS QS system reduced biofilm thickness and <i>P. aeruginosa</i> localization in mono- and polymicrobial biofilms.