A novel in vitro wound biofilm model used to evaluate low-frequency ultrasonic-assisted wound debridement.

OBJECTIVE: Bacterial biofilms remain difficult to treat. The biofilm mode of growth enables bacteria to survive antibiotic treatment and the inflammatory reaction. Low-frequency ultrasound has recently been shown to improve healing in a variety of settings. It is hypothesised that ultrasound disrupts the biofilm leaving bacteria more vulnerable to antiseptic or antibiotic treatment. The objective of this study is to develop a realistic model to elucidate the effect of ultrasound on biofilms. METHOD: A novel in vitro wound biofilm model was developed. Biofilms of Staphylococcus aureus were casted in a semi-solid agar gel composed of either tryptic soy broth (TSB) or a wound simulating media (WSM; composed of Bolton broth with blood and plasma), to resemble the non-surface attached aggregates. The model was used to evaluate the antibiofilm effect of an ultrasonic-assisted wound debridement device (UAW) in the presence of saline irrigation and treatment with a polyhexamethylene biguanide (PHMB)-containing antiseptic. Confocal microscopy was used to evaluate the effect of treatments on biofilm disruption and cell viability counting measured the antibacterial effects. RESULTS: Confocal microscopy showed that application of 10 seconds of moderate-intensity UAW could effectively disrupt semi-solid biofilms grown on both media settings. This treatment only had a small effect on the cell viability. A 24-hour treatment with PHMB was able to reduce the number of bacteria but not eradicate the biofilm in both media settings. Interestingly, the efficacy of the PHMB antiseptic was significantly higher when applied on biofilms grown in the more complex WSM media. However, we found a significant improvement in reducing the number of viable bacteria grown on both media when applying UAW before administration of the PHMB solution. Applying UAW in the presence of PHMB further improved the efficacy. CONCLUSION: Using a realistic in vitro biofilm wound model, we show combining UAW with a PHMB-containing antiseptic has potential as an antibiofilm strategy in wound care. DECLARATION OF INTEREST: The manufacturer of the ultrasonic-assisted wound debridement device, Söring GmbH, Germany, has supported the ultrasound studies. The funding company had no role in the design, data collection, analysis, review, or approval of the manuscript.

Species-specific engagement of human nucleotide oligomerization domain 2 (NOD)2 and Toll-like receptor (TLR) signalling upon intracellular bacterial infection: role of Crohn's associated NOD2 gene variants.

Recognition of bacterial peptidoglycan-derived muramyl-dipeptide (MDP) by nucleotide oligomerization domain 2 (NOD2) induces crucial innate immune responses. Most bacteria carry the N-acetylated form of MDP (A-MDP) in their cell membranes, whereas N-glycolyl MDP (G-MDP) is typical for mycobacteria. Experimental murine studies have reported G-MDP to have a greater NOD2-stimulating capacity than A-MDP. As NOD2 polymorphisms are associated with Crohn's disease (CD), a link has been suggested between mycobacterial infections and CD. Thus, the aim was to investigate if NOD2 responses are dependent upon type of MDP and further to determine the role of NOD2 gene variants for the bacterial recognition in CD. The response pattern to A-MDP, G-MDP, Mycobacterium segmatis (expressing mainly G-MDP) and M. segmatisΔnamH (expressing A-MDP), Listeria monocytogenes (LM) (an A-MDP-containing bacteria) and M. avium paratuberculosis (MAP) (a G-MDP-containing bacteria associated with CD) was investigated in human peripheral blood mononuclear cells (PBMCs). A-MDP and M. segmatisΔnamH induced significantly higher tumour necrosis factor (TNF)-α protein levels in healthy wild-type NOD2 PBMCs compared with G-MDP and M. segmatis. NOD2 mutations resulted in a low tumour necrosis factor (TNF)-α protein secretion following stimulation with LM. Contrary to this, TNF-α levels were unchanged upon MAP stimulation regardless of NOD2 genotype and MAP solely activated NOD2- and Toll-like receptor (TLRs)-pathway with an enhanced production of interleukin (IL)-1ß and IL-10. In conclusion, the results indicate that CD-associated NOD2 deficiencies might affect the response towards a broader array of commensal and pathogenic bacteria expressing A-MDP, whereas they attenuate the role of mycobacteria in the pathogenesis of CD.

Biofilms in wounds: a review of present knowledge.

Following confirmation of the presence of biofilms in chronic wounds, the term biofilm became a buzzword within the wound healing community. For more than a century pathogens have been successfully isolated and identified from wound specimens using techniques that were devised in the nineteenth century by Louis Pasteur and Robert Koch. Although this approach still provides valuable information with which to help diagnose acute infections and to select appropriate antibiotic therapies, it is evident that those organisms isolated from clinical specimens with the conditions normally used in diagnostic laboratories are mainly in a planktonic form that is unrepresentative of the way in which most microbial species exist naturally. Usually microbial species adhere to each other, as well as to living and non-living surfaces, where they form complex communities surrounded by collectively secreted extracellular polymeric substances (EPS). Cells within such aggregations (or biofilms) display varying physiological and metabolic properties that are distinct from those of planktonic cells, and which contribute to their persistence. There are many factors that influence healing in wounds and the discovery of biofilms in chronic wounds has provided new insight into the reasons why. Increased tolerance of biofilms to antimicrobial agents explains the limited efficacy of antimicrobial agents in chronic wounds and illustrates the need to develop new management strategies. This review aims to explain the nature of biofilms, with a view to explaining their impact on wounds.