A three-dimensional bacterial biofilm model for investigating host-biofilm interactions and antibiotic effects

Abstract

Bacterial biofilms are polymicrobial aggregates embedded in a self-produced matrix, which consists of extracellular polymeric substances (EPS). In a clinical context, bacterial biofilms are related to the development of chronic infections. Notably, their unique properties confer persistence in host niches despite antimicrobial treatment and the host immune response, where their distinct virulence exacerbates disease progression. In vitro models for bacterial biofilms have been developed. Simple models typically provide artificial growth environments, resulting in biofilm development diverging from in vivo biofilms. Consequently, sophisticated models incorporate growth conditions mimicking the environment of chronic infections. Still, they lack host cells, as the pathogenic bacteria cause cytotoxic effects during biofilm formation. In this context, electrospinning represents a promising approach, due to the ability to design nanofibrous scaffolds that closely mimic biological matrices, while maintaining mechanical strength for non-destructive handling in in vitro experiments. The objective of this thesis was to develop an in vitro biofilm model for chronic wound infections based on electrospun scaffolds, offering high clinical relevance through its close representation of in vivo conditions, the capacity to explore host-biofilm interfaces, and the incorporation of multiple bacterial species within a single biofilm. Three-dimensional electrospun scaffolds of cellulose acetate (CA) and gelatin were fabricated as scaffolds for biofilm development. The scaffolds were designed to possess composition and physicochemical properties similar to native biofilm microenvironments. Furthermore, high mechanical strength was required to enable the transfer of mature biofilms. The CA-based scaffolds, alone and in combination with gelatin, fulfilled the necessary criteria for physicochemical properties. Gelatin fiber scaffolds were limited due to rapid dissolution in aqueous environments. P. aeruginosa colonization of CA scaffolds did not result in three-dimensional model systems. In contrast, fiber scaffolds of CA and gelatin promoted bacterial distribution throughout their entire dimensions and enabled matrix production. Along with the formation of aggregates and a dense biofilm morphology, this confirmed their maturation. Their high antibiotic tolerance demonstrated the functional properties of mature biofilms. Subsequently, the high mechanical strength of the biofilm models on blend scaffolds enabled the transfer of mature biofilms to ex vivo human skin wounds, imitating a chronic state of wound infection. The novel biofilm model based on nanofibrous scaffolds addressed the major limitations of existing biofilm models by mimicking the native microenvironment of infectious biofilms and facilitating the examination of biofilm-host interfaces. In the next step, the developed biofilm model system was used to evaluate host biofilm interactions. For this study, in vitro and ex vivo models of human skin wounds were infected with pre-cultivated biofilms of P. aeruginosa. Regarding the structural characterization of biofilm infected wound models, all models enabled biofilm growth in close contact to the wound bed, creating ideal conditions for biofilm host interactions. In the case of viable wound models, an active immune response was demonstrated with cytokine gene expression patterns consistent with in vivo observations. Furthermore, the tissue viability was found to impact bacterial colonization behavior in the wound bed. These observations underscored the relevance of using viable models for studying direct biofilm-host-interactions. The new biofilm model, combined with human wound models of various complexities, demonstrated its applicability across a spectrum of research fields, from basic investigations to translational approaches. Biofilms in persistent wound infections typically exhibit a polymicrobial nature. Previous investigations have largely concentrated on single species due to the challenges of co-cultivating biofilms of multiple pathogens in vitro. P. aeruginosa and S. aureus were selected to create mono and dual-species biofilms. P. aeruginosa mono-species biofilms and dual-species biofilms exhibited rapid biofilm development. In contrast, the development of S. aureus biofilms was delayed. Initially, the presence of P. aeruginosa stimulated the adhesion of S. aureus in the dual-species biofilms. However, P. aeruginosa became increasingly dominant, revealing both cooperative and competitive interactions. In dual-species biofilms, both species existed in close proximity, essential for interbacterial interactions. All models exhibited high tolerance to antibiotic treatment. The biofilm matrix acting as a penetration barrier was demonstrated as a major factor. Constituents of P. aeruginosa mono-species and dual-species biofilms impaired wound healing by stimulating host immune responses and reducing cell viability. In contrast, S. aureus biofilms appeared to exhibit proliferative effects with no impairment of wound healing. The biofilm model facilitated the co-cultivation of multiple species in one biofilm, underscoring its similarity to the in vivo environment.

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Identifiers

DOI

10.21248/gups.88139

Journal

Unknown journal

Year

2024