Antimicrobial treatment of methicillin-resistant Staphylococcus pseudintermedius associated with canine wounds represents an important challenge. The aim of this study was to create a canine wound infection model, Lubbock Chronic Wound Biofilm (LCWB), with a focus on S. pseudintermedius, drawing inspiration from the established human model involving S. aureus. Methicillin-resistant S. pseudintermedius 115 (MRSP) and Pseudomonas aeruginosa 700 strains, isolated from dog wounds, were used to set up the LCWB at 24, 48 and 72h. The LCWBs were evaluated in terms of volume, weight, and microbial CFU/mg. The microbial spatial distribution in the LCWBs was assessed by SEM and CLSM imaging. The best incubation time for the LCWB production in terms of volume (3.38 cm3 ± 0.13), weight (0.86 gr ± 0.02) and CFU/mg (up to 7.05 x 106 CFU/mg ± 2.89 x 105) was 48h. The SEM and CLSM images showed a major viable microbial colonization at 48h with a non-mixed bacteria with a prevalence of MRSP on the surface and P. aeruginosa 700 in the depth of the wound. The obtained findings demonstrate the capability of S. pseudintermedius to grow together P. aeruginosa in the LCWB model, representing the suitable model to reproduce the animal chronic wound in vitro.

Chronic wounds have harmful effects on both patients and healthcare systems. Wound chronicity is attributed to an impaired healing process due to several host and local factors that affect healing pathways. The resulting ulcers contain a wide variety of microorganisms that are mostly resistant to antimicrobials and possess the ability to form mono/poly-microbial biofilms. The search for new, effective and safe compounds to handle chronic wounds has come a long way throughout the history of medicine, which has included several studies and trials of conventional treatments. Treatments focus on fighting the microbial colonization that develops in the wound by multidrug resistant pathogens. The development of molecular medicine, especially in antibacterial agents, needs an in vitro model similar to the in vivo chronic wound environment to evaluate the efficacy of antimicrobial agents. The Lubbock chronic wound biofilm (LCWB) model is an in vitro model developed to mimic the pathogen colonization and the biofilm formation of a real chronic wound, and it is suitable to screen the antibacterial activity of innovative compounds. In this review, we focused on the characteristics of chronic wound biofilms and the contribution of the LCWB model both to the study of wound poly-microbial biofilms and as a model for novel treatment strategies.

Innovative non-antibiotic compounds such as graphene oxide (GO) and light-emitting diodes (LEDs) may represent a valid strategy for managing chronic wound infections related to resistant pathogens. This study aimed to evaluate 630 nm LED and 880 nm LED ability to enhance the GO antimicrobial activity against Staphylococcus aureus- and Pseudomonas aeruginosa-resistant strains in a dual-species biofilm in the Lubbock chronic wound biofilm (LCWB) model. The effect of a 630 nm LED, alone or plus 5-aminolevulinic acid (ALAD)-mediated photodynamic therapy (PDT) (ALAD-PDT), or an 880 nm LED on the GO (50 mg/l) action was evaluated by determining the CFU/mg reductions, live/dead analysis, scanning electron microscope observation, and reactive oxygen species assay. Among the LCWBs, the best effect was obtained with GO irradiated with ALAD-PDT, with percentages of CFU/mg reduction up to 78.96% ± 0.21 and 95.17% ± 2.56 for S. aureus and P. aeruginosa, respectively. The microscope images showed a reduction in the cell number and viability when treated with GO + ALAD-PDT. In addition, increased ROS production was detected. No differences were recorded when GO was irradiated with an 880 nm LED versus GO alone. The obtained results suggest that treatment with GO irradiated with ALAD-PDT represents a valid, sustainable strategy to counteract the polymicrobial colonization of chronic wounds.

Resistant wound microorganisms are becoming an extremely serious challenge in the process of treating infected chronic wounds, leading to impaired healing. Thus, additional approaches should be taken into consideration to improve the healing process. The use of natural extracts can represent a valid alternative to treat/control the microbial infections in wounds. This study investigates the antimicrobial/antivirulence effects of Capparis spinose aqueous extract against the main chronic wound pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The extract shows phenolic characterization with rutin (1.8 ± 0.14 μg/mg) as the major compound and antibacterial effect against bacteria (S. aureus PECHA 10 MIC 6.25%; P. aeruginosa PECHA 4 MIC 12.50%) without action against C. albicans (MIC and MFC ≥ 50%). Capparis spinose also shows a significant antivirulence effect in terms of antimotility/antibiofilm actions. In particular, the extract acts (i) on P. aeruginosa both increasing its swimming and swarming motility favoring the planktonic phenotype and reducing its adhesive capability, (ii) on S. aureus and P. aeruginosa biofilm formation reducing both the biomass and CFU/ml. Furthermore, the extract significantly displays the reduction of a dual-species S. aureus and P. aeruginosa Lubbock chronic wound biofilm, a complex model that mimics the realistic in vivo microbial spatial distribution in wounds. The results suggest that C. spinose aqueous extract could represent an innovative eco-friendly strategy to prevent/control the wound microbial infection.

Chronic wound infections represent an important health problem due to the reduced response to antimicrobial treatment of the pathogens organized in structured biofilms. This study investigated the effects of the previously described antifungal peptide L18R against three representative wound pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The antimicrobial activity of L18R was evaluated (i) against single planktonic microbial populations; (ii) on single, dual, and triadic species of biofilms in both the early stage and mature stage; and (iii) in the polymicrobial Lubbock chronic wound biofilm (LCWB) model, mimicking spatial microbial colonization. This study used the evaluation of CFUs, biofilm biomass detection, and confocal and scanning electron microscopy analysis. L18R showed a significant antimicrobial activity against planktonic microorganisms and was able to differentially reduce the biomass of monomicrobial biofilms. No reduction of biomass was observed against the polymicrobial biofilm. In mature LCWB, L18R caused a moderate reduction in total CFU number, with a variable effect on the different microorganisms. Microscopy images confirmed a predominant presence of P.aeruginosa and a lower percentage of C. albicans cells. These findings suggest a modulating action of L18R and recommend further studies on its potential role in chronic wound management in association with conventional antibiotics or alternative treatments.