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.
Highlights
Skin is the largest organ of the body and functions as a physical barrier against external insults, such as toxins or pathogenic microorganisms (Parlet et al, 2019)
The results obtained indicate that the model offers significant potential as a screening platform to assess interventions that reduce pathogen biofilms, promote healthy skin or aid healing as well as for investigating interactions that occur between commensals, pathogens and host keratinocytes (Egert and Simmering, 2016)
In co-cultures where P. aeruginosa has been reported to outcompete or largely inhibit staphylococcal growth (S. aureus or S. epidermidis), comparable numbers of both bacterial species co-inoculated at the same time have generally been used (Hotterbeekx et al, 2017; Bahamondez-Canas et al, 2019)
Summary
Skin is the largest organ of the body and functions as a physical barrier against external insults, such as toxins or pathogenic microorganisms (Parlet et al, 2019). Skin is principally composed of an epidermis and underlying dermis with keratinocytes constituting 90–95% of the upper epidermal layer nearest to the colonizing microbiota. The stratum corneum is a waxy waterproof composite containing flattened corneocytes and interlocking keratinocyte-derived lipids and granules that forms a tight mechanical barrier Despite this seemingly inhospitable niche that is acidic, lipid dense and lacking in nutrients, direct contact with the environment results in skin becoming colonized by a diverse community of bacteria, fungi, viruses and mites, for which the skin offers a variety of stable niches with different environmental conditions and nutrients (Grice and Segre, 2011; Brandwein et al, 2016; Byrd et al, 2018; Erin Chen et al, 2018). Staphylococcus epidermidis autoinducing peptide (AIP) signal molecules inhibit the Staphylococcus aureus agr system and exotoxin production (Otto et al, 2001) whereas Microccus luteus enhances S. aureus pathogenesis (Boldock et al, 2018)
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