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].…”

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].…”

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, the 4-D microenvironment supports the co-existence of P. aeruginosa and S. aureus in mixed-species biofilms, with a relative predominance of P. aeruginosa . This aligns with previous in vivo and clinical observations of wound biofilms [ 6 , 7 , 18 , 23 , 35 , 100 ], as well as biomimetic in vitro studies, where P. aeruginosa and S. aureus co-exist in wound-like conditions, however P. aeruginosa is found in larger numbers [ 18 , 35 ]. This is possibly due to the inhibitory effect of P. aeruginosa on the growth of S. aureus (widely observed under in vitro conditions) [ 18 , 21 , 99 ], but could also be due to a combination of factors, including the increased growth of P. aeruginosa under mixed-species conditions [ 23 ].…”

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

“…The treatment of the P. aeruginosa 3D biofilm model with the wound dressings for 24 h resulted in a significant reduction in the bacterial load with all antimicrobial wound dressings compared to the wound dressings without active additives. These results are consistent with previous investigations [ 24 , 43 , 58 , 61 ]. A comparable antimicrobial effect was observed using the PHMB containing wound dressing and the dressing with ionic silver.…”

“…PHMB interacts with negatively charged phospholipids in the bacterial membranes leading to loss of integrity followed by cell death [ 54 ]. The observed antimicrobial effects of PHMB dressings in our established multiple species biofilm model are in accordance with previous in vitro investigations [ 42 , 43 , 53 , 55 , 58 , 59 ].…”

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].…”

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, the 4-D microenvironment supports the co-existence of P. aeruginosa and S. aureus in mixed-species biofilms, with a relative predominance of P. aeruginosa . This aligns with previous in vivo and clinical observations of wound biofilms [ 6 , 7 , 18 , 23 , 35 , 100 ], as well as biomimetic in vitro studies, where P. aeruginosa and S. aureus co-exist in wound-like conditions, however P. aeruginosa is found in larger numbers [ 18 , 35 ]. This is possibly due to the inhibitory effect of P. aeruginosa on the growth of S. aureus (widely observed under in vitro conditions) [ 18 , 21 , 99 ], but could also be due to a combination of factors, including the increased growth of P. aeruginosa under mixed-species conditions [ 23 ].…”

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

“…The treatment of the P. aeruginosa 3D biofilm model with the wound dressings for 24 h resulted in a significant reduction in the bacterial load with all antimicrobial wound dressings compared to the wound dressings without active additives. These results are consistent with previous investigations [ 24 , 43 , 58 , 61 ]. A comparable antimicrobial effect was observed using the PHMB containing wound dressing and the dressing with ionic silver.…”

“…PHMB interacts with negatively charged phospholipids in the bacterial membranes leading to loss of integrity followed by cell death [ 54 ]. The observed antimicrobial effects of PHMB dressings in our established multiple species biofilm model are in accordance with previous in vitro investigations [ 42 , 43 , 53 , 55 , 58 , 59 ].…”

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].…”

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, the 4-D microenvironment supports the co-existence of P. aeruginosa and S. aureus in mixed-species biofilms, with a relative predominance of P. aeruginosa . This aligns with previous in vivo and clinical observations of wound biofilms [ 6 , 7 , 18 , 23 , 35 , 100 ], as well as biomimetic in vitro studies, where P. aeruginosa and S. aureus co-exist in wound-like conditions, however P. aeruginosa is found in larger numbers [ 18 , 35 ]. This is possibly due to the inhibitory effect of P. aeruginosa on the growth of S. aureus (widely observed under in vitro conditions) [ 18 , 21 , 99 ], but could also be due to a combination of factors, including the increased growth of P. aeruginosa under mixed-species conditions [ 23 ].…”

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

“…The treatment of the P. aeruginosa 3D biofilm model with the wound dressings for 24 h resulted in a significant reduction in the bacterial load with all antimicrobial wound dressings compared to the wound dressings without active additives. These results are consistent with previous investigations [ 24 , 43 , 58 , 61 ]. A comparable antimicrobial effect was observed using the PHMB containing wound dressing and the dressing with ionic silver.…”

“…PHMB interacts with negatively charged phospholipids in the bacterial membranes leading to loss of integrity followed by cell death [ 54 ]. The observed antimicrobial effects of PHMB dressings in our established multiple species biofilm model are in accordance with previous in vitro investigations [ 42 , 43 , 53 , 55 , 58 , 59 ].…”