A novel chronic wound biofilm model sustaining coexistence of Pseudomonas aeruginosa and Staphylococcus aureus suitable for testing of antibiofilm effect of antimicrobial solutions and wound dressings

Abstract

Chronic wounds are a large burden to patients and healthcare systems. Biofilm infections in chronic wounds are crucial factors leading to non-healing of wounds. It is important to study biofilm in wounds and to develop effective interventions against wound biofilm. This study presents a novel in vitro biofilm model mimicking infected chronic wounds. The novel layered chronic wound biofilm model uses woundlike media and includes both Pseudomonas aeruginosa and Staphylococcus aureus, which have been identified as the most important pathogens in wounds. The model sustains their coexistence for at least 96 h. Microscopy of the model revealed microbial growth in non-surface attached microcolonies as previously observed in vivo. The model was used to determine log 10 -reduction for the use of an antimicrobial solution and antimicrobial dressings (containing silver or honey) showing moderate-to-low antibiofilm effect, which indicates better concordance with the observed clinical performance of this type of treatment than other widely used standard tests. K E Y W O R D S biofilm, coexistence of Pseudomonas aeruginosa and Staphylococcus aureus, model, test of antibiofilm effect | INTRODUCTIONChronic wounds constitute a large burden on patients, society and healthcare systems, affecting 2% of the population in the developed countries with an estimated 2%-3% of the healthcare budgets related to chronic wounds globally, equalling US $13-15 billion annually. [1][2][3] Bacteria inflict on the healing of chronic wounds, 4 with Pseudomonas aeruginosa being identified as a major pathogen 5 together with Staphylococcus aureus, but the underlying mechanisms on the pathogenicity of wound biofilm are not well understood. 6 Furthermore, the Chen and Lorenzen served as co-first authors.

Adding a new dimension: Multi-level structure and organization of mixed-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a 4-D wound microenvironment

“…Taken together, this indicates that the 4-D microenvironment supports the co-existence of P. aeruginosa and S. aureus in mixed-species biofilms, with P. aeruginosa steadily outcompeting S. aureus . This aligns with previous in vivo and clinical observations of wound biofilms [6,7,18,21,33,71], as well as biomimetic in vitro studies, where P. aeruginosa and S. aureus are seen to co-exist in wound-like conditions, with P. aeruginosa found in larger numbers [18,33]. The predominance of P. aeruginosa in the mixed-species biomass could be due to an inhibitory effect of P. aeruginosa on the growth of S. aureus [18,72,73] (widely observed under in vitro conditions), or due to the inherent growth dynamics of the individual bacterial species in the 4-D microenvironment, such as the increased growth of P. aeruginosa under mixed-species conditions [21].…”

3D Biofilm Models Containing Multiple Species for Antimicrobial Testing of Wound Dressings

“…The spatial nature of the models allows for good transferability to the in vivo situation during antimicrobial treatment with wound dressings, as effects on biofilm not only in direct contact, but also in deep tissue, can be recorded. So far, there are only a few in vitro biofilm models not based on surface attached biofilms growing in clusters [ 31 , 41 , 42 , 43 ]…”

“…In recent years, the need to develop biofilm models with more than one bacterial species has been recognized. Models exist that incorporate two different bacteria, e.g., S. aureus and P. aeruginosa in a multi-layered agar model [ 43 ], in a cellulose on agar model [ 44 ], or in a coagulated Wound-Like Medium model [ 45 ]. Furthermore, biofilm models with three [ 46 , 47 , 48 ], four [ 31 ], or five bacteria species [ 44 ] were established.…”

Saudi Honey: A Promising Therapeutic Agent for Treating Wound Infections

“…Infection with S. pyogenes is always considered of high concern due to its serious damage to the skin, known as necrotizing fasciitis [7], while both S. aureus and S. pyogenes have certain virulence factors (e.g., hemolysins) that are associated with impaired wound healing [8,9]. Wound pathogens, including P. aeruginosa and S. aureus, are well-known for their ability to form biofilms through the use of surface proteins (e.g., protein F1/SfbI), which adhere to the host cells and enable bacterial aggregation [10]. Biofilms exhibit considerable resistance to antibiotics, which may impair wound healing and deteriorate the clinical outcome [10].…”

“…Wound pathogens, including P. aeruginosa and S. aureus, are well-known for their ability to form biofilms through the use of surface proteins (e.g., protein F1/SfbI), which adhere to the host cells and enable bacterial aggregation [10]. Biofilms exhibit considerable resistance to antibiotics, which may impair wound healing and deteriorate the clinical outcome [10].…”