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 in vitro 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 (Staphylococcus epidermidis and Micrococcus luteus) and pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) 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 P. aeruginosa and S. aureus, much thinner biofilms were observed than those formed by the pathogens alone. When P. aeruginosa was inoculated with S. epidermidis in the presence or absence of M. luteus, the commensals formed a layer between the keratinocytes and pathogen. Although S. aureus completely inhibited the growth of M. luteus in dual-species biofilms, inclusion of S. epidermidis in triple or quadruple species biofilms, enabled M. luteus to retain viability. Using this polymicrobial biofilm keratinocyte model, we demonstrate that a quorum sensing (QS) deficient S. aureus agr 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 P. aeruginosa localization in mono-and polymicrobial biofilms.

Challenges in exploring and manipulating the human skin microbiome

“…Polymicrobial biofilms formed by a mixture of commensal strains ( Staphylococcus epidermidis and Micrococcus luteus ) and pathogens ( Staphylococcus aureus and Pseudomonas aeruginosa ) were also used to study the interactions among commensal organisms, pathogens, and human keratinocytes. The authors observed that the commensal organisms reduced the damage caused to the keratinocyte monolayer by pathogens, reduced biofilm thickness, and formed a layer between the keratinocytes and pathogens [ 117 ] .…”

Investigating pathogenicity and virulence of Staphylococcus pettenkoferi: an emerging pathogen

“…To test this hypothesis, human skin keratinocytes were used to evaluate the survival capacity of S. pettenkoferi SP165. HaCaT is a non-transformed human keratinocyte line that has been used to investigate epithelial infection 25 , cytotoxicity 26 , biofilm formation 27 , and skin cancer 28 . It can also be employed in models of skin wound infection where the stratum corneum is disrupted and bacteria come into close contact with living keratinocytes 27 .…”

“…HaCaT is a non-transformed human keratinocyte line that has been used to investigate epithelial infection 25 , cytotoxicity 26 , biofilm formation 27 , and skin cancer 28 . It can also be employed in models of skin wound infection where the stratum corneum is disrupted and bacteria come into close contact with living keratinocytes 27 . Therefore, we performed invasion experiments to address the persistence of S. pettenkoferi SP165 since S. aureus has been demonstrated to proliferate and persist intracellularly in HaCaT cells 29,30 .…”

Ignoring social distancing: advances in understanding multi-species bacterial interactions

“…Furthermore, using transcriptomes, several studies have shown that bacterial behavior in the laboratory is not consistent with the behavior in a natural in situ environment 38 , 39 . Aiming to more accurately replicate native environments, several new model systems have been developed, including artificial urine media 40 for an investigation into polymicrobial urinary tract infections, complex skin organoids for skin-community colonization 41 , the inclusion of food-grade fibers in a gut model 42 , as well as several systems facilitating cultivation in native environments 15 , 16 . These models, based on researcher rationale, are likely superior models, but without validation it is unclear how they compare to the native environment.…”